Technical

RNA Sample Preparation Guide
source:Technical divisiondate:2022-06-08views:754

1RNA SAMPLE yield and minimum input amount for various projects

Total RNA yield statistics of each sample (for reference only)

Recommended starting amount for sequencing projectsthe starting amount is for reference only,exosomes do not allow 18/s28s rRNA peaks):

Expression Gene Chip and Small RNA Sequencing Body Fluid Samples and Exosome Project Starting Amount (for reference only)

 

2Various sample preparation guides

2.1 Animal tissue/clinical tissue collection

Take fresh tisse,the vollume of the tissue should be small,and the length,width and height should be≤0.5cm as far as possible.Considering the operability, the size of the cut tissue block can refer to the size of mung bean particles.

② To remove non-researched tissue types (such as connective tissue, adipose tissue, etc.), if it is a clinical lesion tissue to correctly determine the lesion and normal tissue, the normal tissue around the lesion tissue should be removed, and vice versa.

 

③ Immediately wash the blood and stains on the tissue surface with 1×PBS or normal saline prepared with RNase-Free water pre-cooled at 2-6℃, and dry the liquid on the surface with a lint-free paper towel.

④ The treated tissue was quickly put into a pre-cooled RNase-Free threaded port with a threaded mouth and was cryogenically resistant to -192 °C, and quickly placed in liquid nitrogen for 1 hour, and then transferred to -80 °C for long-term storage. Store and avoid repeated freezing and thawing prior to RNA extraction.

Precautions: 

  1. If the laboratory does not have the conditions, such as the lack of liquid nitrogen, dry ice and -80°C refrigerator, samples that require RNAlater and similar tissue preservation solutions should be stored in strict accordance with the operating instructions, and the samples should be quickly divided into lengths, widths and heights ≤0.5 cm, a small piece about the size of a pea (usually weighs about 100mg, but does not require accurate measurement, generally requires about 10 small pieces), because the tissue block will affect the efficiency of RNAlater penetration into the tissue, and the tissue block will also cause RNAlater. The tissue cannot be completely covered, and the tissue parts not covered by RNAlater are highly susceptible to degradation by nucleases. Then put it into a 1.5mL EP tube filled with RNAlater prepared in advance, make the sample completely immersed in the liquid, and then store it at 4°C overnight, transport it on dry ice, or store it at -80°C.
  2. It is not allowed to send tissue samples preserved with lysis buffer, because the quality of RNA extracted from tissue samples preserved with Trizol, whether it is ground powder or cut tissue blocks, is generally poor;
  3. Animals and clinical tissues should not be wrapped in tinfoil. The tinfoil is easy to stick with animals and clinical tissues, and the tinfoil is easy to be brought in during the RNA extraction process, causing pollution.

 

2.2 Collection of cell samples

Handling of adherent cells

① Take out the adherent growing cells from the incubator, observe the cells under a microscope to confirm that the growth state is good, discard the medium,

Wash the cells by laying flat for 1 min after adding 1×PBS prepared with the same volume of enzyme-free water as the culture medium,then discard the PBS and repeat once.

③ Add an appropriate amount of lysis solution and repeatedly pipette until fully lysed (Take TriZol as an example, a 10-15cm large dish, add 1-2mL each time, pipetting and mixing, the standard for sufficient lysis is that the liquid is not viscous and has good fluidity when pipetting. If the liquid is too viscous, it means that the lysate has not been fully lysed. It is necessary to add lysis solution. The recommended amount of each addition is about 100μL and continue to add until the liquid is not viscous)

Store at -80℃ refrigerator for long-term preservation after transferring to -192℃low temperature threaded cryopreservation tube. Transported on dry ice.

 

Handling of suspension cells

Select cell suspensions with good growth conditions.

Centrifuge at 200-1000g for 5-10min to obtain cell pellet and discard the medium.

After adding an appropriate amount of 1xPBS prepared with RNase-Free water,resuspend the cell pellet by gently pipetting with a wide-mouth pipette tip,then centrifuge at 200g for 5min,discard the PBS,repeat once.

④Add an appropriate amount of TriZol lysis solution and pipet repeatedly until fully lysed (the lysis standard is similar to the treatment method for adherent cells above)

Store at -80℃ refrigerator for long-term preservation after transferring to -192℃low temperature threaded cryopreservation tube. Transported on dry ice.

Precautions

  1. Do not send frozen cells directly, because ice crystals can easily pierce the cells during the freezing process, and the broken cells will easily degrade RNA and RNA.

b. Do not digest cell samples with trypsin. Trypsin will digest membrane proteins on cell membranes, causing cells to rupture, and ruptured cells will release nucleases.

2.3 Plant tissue collection

①Select fresh, tender, and vigorously growing tissue parts. The younger and fresher the plant tissue, the less secondary metabolites it contains. Secondary metabolites can affect RNA extraction. Take the strawberry flower as an example. For such a size of flower, 4 flowers are enough, and so on for other flower samples.

②(Optional for some samples) Quickly wash the stains on the surface of the tissue with 1×PBS or normal saline prepared with pre-cooled RNase-Free water, and blot the liquid on the surface. Cut it into a suitable size with scissors (if it is not a special case, the length should not exceed 2cm)

Put the processed tissue into a pre-cooled RNase-Free threaded cryopreservation tube with a number of -192°C immediately , or wrap it in tin foil with a marked name, and freeze it in liquid nitrogen for > 1 hour. Transfer to -80℃ for long-term storage, avoid repeated freezing and thawing before RNA extraction, and send samples on dry ice. If the liquid nitrogen is not used for quick freezing and cooling within 10 minutes after separation, it is very likely that the sample will degrade. Do not store the sample directly at -80°C. At this time, RNase with low activity can still cause sample degradation.

Precautions

a. Plant tissue is not recommended for RNAlater preservation, because plant tissue contains cell walls and secondary walls, and RNAlater is not easy to penetrate into the tissue; especially for plants with waxy surfaces, do not directly soak plant tissue with TriZol

b. Plant tissue is not allowed to be ground into powder for delivery.

c. Plant samples containing more wax on the surface or plant tissues with extremely high content of secondary metabolites or stems, roots and seeds of plant tissues usually have lower RNA yields. In order to improve the yield of RNA, it is necessary to increase the sample volume, which is 3-4 times the sample volume of ordinary samples.

2.4 Whole blood/PBMC/serum/plasma and other body fluid sample collection

Lianchuan Bio recommends using BD's PAXgene™ blood collection tube to collect whole blood, BD's vacuum blood collection tube to collect whole blood and then separate serum, or use EDTA purple anticoagulant tube to collect whole blood samples and separate subsequent plasma samples. Whole blood collection with green heparin tubes is not allowed because heparin affects the reverse transcription efficiency of the PCR enzyme.

a. The way of whole blood collection with PAXgene lood collection tubes

①Before blood collection, the blood collection tube should be returned to room temperature. The recommended temperature is 18-25°C (generally, PAXgene blood collection tubes are stored at room temperature or 4°C, and the maximum temperature does not exceed 40°C).

②When collecting blood, if a variety of blood collection tubes have been used to collect blood, the PAXgene blood collection tube should be used at the end; when only PAXgene blood collection tubes are used, first use another blood collection tube to collect 1-2 mL blood samples, discard the blood collection tube, and then use PAXgene blood collection tubes. This effect is because the volume of the blood collection device can be pre-perfused, and the impact force caused by blood pressure is reduced, and the blood collection tube is cleaned to a certain extent. Blood collection volume: 2.5ml, applicable objects: humans and primates.

After blood collection, invert up and down for about ten times immediately, and place it upright at 18-25°C for 2 hours.

④ If it is not extracted immediately, place the blood collection tube upright on the plastic test tube rack (note: there should be a gap between the test tube rack and the blood collection tube to prevent the tube wall from cracking during cryopreservation). Then transfer to -80°C for long-term storage or to dry ice for shipping. Note that in this step, the whole blood sample in the blood collection tube needs to be transferred to a threaded cryogenic tube and placed in a -80°C freezer. There is no need to quick-freeze the cryogenic tube in liquid nitrogen for 1 hour.

⑤Please use a thick-walled foam box for transportation and put enough dry ice to avoid repeated freezing and thawing during sample storage and transportation.

b. The way of EDTA blood collection tube/vacuum blood collection tube to collect whole blood

Use 75% alcohol cotton to disinfect the blood collection site from the inside to the surrounding area before sample collection,

②Blood is drawn into a specific blood collection tube, and the quantity reaches the maximum allowable negative pressure,

Mix by gentle inversion 10 times immediately after sample collection. Move as gently as possible. Add more than three times the volume of TriZol (TriZol:blood ≥3:1) lysis solution and mix well. It is normal for precipitation to occur at this time.

Transfer it to a threaded cryopreservation tube with a low temperature of -192°C immediately after mixing, transfer it to a -80°C refrigerator for storage, and transport it on dry ice. Note that the longest time from blood collection to RNA extraction should not exceed one week.

c. PBMCextraction method in plasma

①Following the previous step of centrifuging the EDTA anticoagulant tube and layering, the Ficoll and PBS were returned from low temperature to room temperature.

②Add 10mL Ficoll to a 50mL centrifuge tube (labeled A),then it best to let it stand for a while,Add 10 mL of PBS to another 50 mL centrifuge tube (labeled B)

③Dilution: Gently shake the anticoagulant blood collection tube, remove the cap with kimwipes or sterile gauze and set aside, transfer 10mL of blood to the B centrifuge tube, mix gently to obtain a PBS mixture (20mL)

④Properly tilt the centrifuge tube A, and slowly add 20mL of PBS mixture along the wall (be careful to be slow, too fast will break through the Ficoll layer, which will greatly affect the extraction effect)

⑤Gradient centrifugation: Carefully put the centrifuge tube into the centrifuge (do not disturb the liquid layer), centrifuge at 400g for 20min at room temperature, and set the acceleration/deceleration to 1/0 respectively

⑥After centrifugation, take it out carefully and place it on the operating table. It can be seen that it is divided into four layers, as shown in the figure above.

⑦Optional: first remove 2-3mm of plasma on the lymphocyte layer to facilitate later aspiration

⑧Aspirate PBMC as much as possible with a P1000 pipette, transfer to a 15mL centrifuge tube, try to avoid aspirating plasma and Ficoll

⑨Add PBS to make the volume to 10mL, cap and shake 5 times by gentle inversion

⑩Centrifuge at 300g for 10min at room temperature, with acceleration/deceleration set to 9/9 respectively. Remove the supernatant and flick the end of the centrifuge tube until the cell pellet is completely resuspended in the remaining PBS

PrecautionsRepeat steps ⑨-⑩, and finally add TriZol according to the ratio of TriZol and volume of about 3:1 to ensure full lysis. For lysis standards, please refer to the treatment method of adherent cell lysis solution above.

d. Serum sample(for exosomes)

①It is recommended to use imported medical serum tubes or vacuum blood collection tubes to collect whole blood

Place the whole blood upright at 4℃ or in an ice box immediately after inverting and mixing gently ten times.

③Centrifuge at 1800g for 10min to obtain the upper serum sample (whole blood needs to be separated for serum within 1h, according to the operation instructions of the serum tube or the serum separation procedure of the customer laboratory)

④Transfer the serum to a 1.5mL centrifuge tube and centrifuge at 13000g for 2min;

⑤Transfer the supernatant to a 200μL-1.5mL cryotube with a -192°C ultra-low temperature resistant screw top, and each sample is at least ≥100μL (0.1mL). Quick-freeze in liquid nitrogen for 1 hour, store at -80°C, and transport on dry ice.

e.Plasma samples(for exosomes)

①It is recommended to use purple EDTA anticoagulant tubes to collect whole blood samples

Place the whole blood upright at 4℃ or in an ice box immediately after inverting and mixing gently ten times.

③Centrifuge at 1200g for 10min to obtain the upper plasma sample (whole blood should be separated within 1h, according to the operation instructions of the anticoagulation tube or the plasma separation steps of the customer's laboratory)

④Transfer the plasma to a 1.5mL centrifuge tube and centrifuge at 13000g for 2min

⑤Transfer the supernatant to a 200μL-1.5mL cryotube with a -192°C ultra-low temperature resistant screw top, and each sample is at least ≥100μL (0.1mL). Quick-freeze in liquid nitrogen for 1 hour, store at -80°C, and transport on dry ice

Precautions

a.The separation of serum and plasma should be carried out in strict accordance with the operating instructions. The operation should be completed within 1 hour to avoid hemolysis. Repeated freezing and thawing should be avoided after separation of serum and plasma.

b. The principle of serum plasma sample volume: the more the better, if the customer has less serum plasma on hand, they can send as much as they have. Usually, the RNA content of this type of sample is low, so it is necessary to increase the sample volume to increase the sample size. Enrich enough RNA to ensure the smooth progress of subsequent experiments, in addition, serum plasma RNA is usually not quality checked

c.Whole blood for separating serum/plasma samples should not be frozen, and the mixed blood cell samples should be discarded as much as possible and cannot be used.

D.Serum/plasma RNA yield of 2-8 ng/mL

Tips——

 

Explanation of hemolysis: that is, the rupture of red blood cells and the overflow of hemoglobin caused by the rupture of red blood cell membranes. Hemolysis can be caused by a variety of physical or chemical factors, especially outside the human body, changes in osmotic pressure, external mechanical forces, changes in temperature, changes in pH, or some artificially added chemicals during blood processing may cause varying degrees of hemolysis.

Preventive solution of hemolysis

  1. After the blood leaves the human body, the stability of the blood environment is inversely proportional to the length of storage time, and the stability of the cell membrane is also declining. In this case, the separation of serum and plasma should be carried out as soon as possible; (2) Different anticoagulants have different molecular configurations, which will have different effects on the cell membrane of each cell in the blood, some will increase the stability of the cell membrane, and some will reduce the stability of the cell membrane, so choose an anticoagulant. An appropriate anticoagulant is essential (recommended anticoagulants: EDTA, sodium citrate);

(3) High temperature or repeated freezing and thawing during blood storage will cause great damage to cell membranes. Excessive temperature and repeated freezing and thawing will change the conformation of structural proteins on the cell membrane, which will reduce the elasticity of the cell membrane and cause hemolysis.

f. Cell supernatant, urine, saliva, amniotic fluid, cerebrospinal fluid (similar to exosomes)

①Collect cell supernatant, fresh urine, saliva, amniotic fluid, and cerebrospinal fluid (the starting amount is as indicated in the previous table), making sure not to be hemolyzed (the protein in the blood will contaminate the sample protein); keep it upright at 4°C or in an ice box Set aside and transfer to RNase-free centrifuge tubes. The following operations should be performed within 1 hour after sample collection to prevent RNA degradation

Centrifuge 3000g for about 10min at 4℃ to remove cells and debris

Pour the supernatant carefully into a new RNase-free centrifuge tube (be careful not to transfer the pellet), and centrifuge at 13000g for about 2 min at 4°C to remove residual debris and cells

④Transfer the supernatant to a brand-new -192°C ultra-low temperature-resistant threaded cryopreservation tube, quick-freeze in liquid nitrogen for 1 hour (optional), and seal tightly. Store at -80°C and ship on dry ice.

PrecautionsThe cell supernatant is too fragmented and needs to be centrifuged several times at low speed to remove the debris. The day before urine collection should be light, mid-morning urine (clinical), and 1-hour morning urine (animal). Before saliva collection, you need to fast for more than 2 hours, take samples from 9:30-11:30 in the morning, rinse your mouth with distilled water, spit saliva in a sterile sputum cup (keep a water bath), do not expect sputum, and collect saliva for about 30 minutes. 10000rpm at 4°C for 10min, take the supernatant, filter and sterilize through a 0.22μm filter.

 

2.5 Paraffin tissue

Paraffin block of surgical/biopsy tissue 

A single extraction of RNA requires more than 35 mg. The total amount of surgical tissue excised from the pathology department of the hospital is ≥35 mg (mung bean size, and the section of the tissue exposed to the air is not acceptable). organize. Unsectioned paraffin blocks can be shipped at 4°C or at room temperature.

 

Surgery/Biopsy Paraffin Rolls/Biopsy Paraffin Sections RNA extraction <80 μm in one extraction. Cut paraffin rolls with a tissue thickness of about 10-20 μm from the pathology department of the hospital, 10 pieces of surgical tissue (tissue>1×1cm2), or 20 pieces (tissue<1×1cm2), and 20-25 pieces of punctured tissue. Place the cut rolls in -192°C ultra-low temperature threaded cryopreservation tubes, quickly immerse them in liquid nitrogen for quick freezing for >1h, and store at -80°C or transport them on dry ice.

Precautions

a. Priority order of sample delivery: Paraffin block > paraffin roll > paraffin section

b. All paraffin tissue samples should be submitted for inspection within one year and no more than 18 months. It is recommended to provide an immunohistochemical or histochemical staining section

c. Freshly made slices should be placed within 1 week at room temperature, within 1 month at 2-8°C, and within 6 months at -20 

d. It is strongly recommended to store only one patient's sample in each patch box; if it needs to be placed with other patient samples, it must be separated to prevent contamination caused by sample contact.

e. It is strongly not recommended to use corrugated paper or self-folding Z-shaped paper to place paraffin sections, because the collision during transportation will cause the paraffin sections to squeeze together, resulting in section damage or sample contamination.

 

Paraffin tissue container

Paraffin section example     

2.6 Non-pathogenic bacteria sample collection

The amount of bacteria required for one reaction is ≤1×107

①Observe the growth state under the microscope and collect the logarithmic growth phase bacteria;

②Transfer an appropriate volume of bacterial solution to a 2mL screw-cap RNase-Free centrifuge tube with a pointed bottom, and centrifuge at 14,000g for 4-10min at room temperature or 4°C

③Discard the medium, quickly freeze the bacterial pellet in liquid nitrogen for >1h, then transfer it to -80°C or liquid nitrogen for long-term storage, and transport it on dry ice

2.7 Non-pathogenic fungi sample collection

The amount of fungi required for one reaction is less than or equal to 50 mg, the fungi are weighed and divided into multiple tubes; the weighed fungi are placed in a pre-cooled -192°C ultra-low temperature resistant thread cryopreservation tube, quickly put into liquid nitrogen, and the quick-freezing time > 1h, transfer to -80°C for long-term storage or send directly on dry ice.

2.8 Pathogenic fungi and bacteria treatment methods

All potentially pathogenic fungal and bacterial RNA project samples need to be pretreated according to the following steps before they can be sent to the company, and the laboratory needs to be notified in advance to clarify the bacteria species. Solutions A, B can be requested from the laboratory. The laboratory will destroy untreated samples that may cause disease

①Bacterial/fungal pellet (not more than 30mg, about the size of mung bean) was added to 400ul of solution A, and the bacteria were resuspended by pipetting.

②Add 400ul of solution B to the resuspended cells, shake vigorously and mix.

③Heat in a water bath or metal bath at 65°C for 30 minutes, and shake vigorously every 5 minutes to prevent stratification.

④After the treatment, it was frozen at -80°C and transported on dry ice. If it is pathogenic bacteria, please treat the surface of the container with 70% ethanol (note that if the number is blurred during the processing, rewrite the number after processing) to ensure safety.

Precautions

  1. When the room temperature is low, solution A may precipitate out, so it can be preheated at 65°C for 2-3 minutes to dissolve the precipitation and then mix well.
  2. Solution B is corrosive, please pay attention to safe operation. Solution B is an organic solution, and the upper liquid is a water seal. When pipetting, make sure to insert the pipette tip into the lower liquid.3Solutions A and B are immiscible, and they need to be shaken sufficiently during operation to make the two liquids form an emulsion.

4Please indicate on the sample submission form: the sample is pretreated with solution A and solution B.

2.9 Total RNA sample

Total RNA generally is stored in a -80°C refrigerator for a long time. If you want to send it, you can send it directly on dry ice, or add an appropriate amount of preservation solution, and then transport it on dry ice.

RNA storage medium (recommended concentration >50ng/μL):

①RNA preservation solution (1/10 volume 3M NaAc, pH=5.2, 3 times volume absolute ethanol)

②3 volumes of absolute ethanol

③RNase-free watersample>15μL

Precautions

  1. Residues of impurities such as polysaccharides, proteins, and DNA should be avoided as far as possible in total RNA, and the solvent composition should be indicated when sending samples

(2) Total RNA should avoid repeated freezing and thawing. Since the shear force generated by repeated freezing and thawing will damage the RNA sample, in practice, the RNA sample should be packaged in small quantities.

Auxiliary materials

1. Sample Packaging Notes

①Use an oil-based marker to mark the sample number and sample type on the surface of the aluminum foil or cryopreservation tube that wraps the sample. And do not contact with organic solvents such as ethanol

②Do not wrap the sample directly with gauze or paper, but wrap it in aluminum foil or cryopreservation tube and put it in the sample bag

③Do not store samples in liquid nitrogen in glass containers

④Do not put label paper or other paper-based explanatory documents in the sample bag, as paper is fragile in liquid nitrogen

⑤The label paper should be attached to the sample bag string, not to the surface of the container to avoid falling off and causing sample confusion

⑥ Do not put too many samples in one sample bag in case the liquid nitrogen tank cannot be placed or removed from the liquid nitrogen tank. Sample bags are pre-cooled in liquid nitrogen prior to use

⑦ When filling in the "Sample Registration Form", the customer should describe in detail (for clinical samples, it is better to indicate the clinical etiology and stage) of the sample type, processing method, storage conditions, storage time and other relevant details, so that the technician can determine a reasonable Experimental program

2,Sample Storage Considerations

 After quick freezing in liquid nitrogen, the cryovials or tin foil that carry the samples should be stored at -80°C immediately. Routine samples must be snap-frozen in liquid nitrogen immediately within 10 minutes, and the samples cannot be directly stored at -80°C without liquid nitrogen quick-freezing treatment. Samples that have not been quenched with liquid nitrogen can also remain in low activity enzymes, causing nucleic acid degradation and protein denaturation

② After the tissue sample is collected, the sample should be put into an imported high-quality cryogenic tube quickly. It is recommended to use an imported cryogenic tube with a low temperature of -192°C, and put it into liquid nitrogen immediately after tightening it. Then transfer to -80°C for storage. If it is not tightened, liquid nitrogen will flow into the freezing tube, and the air will explode after thermal expansion and contraction.

③ Tissue samples were stored in liquid nitrogen or -80°C freezer. For samples stored in liquid nitrogen, be careful not to use non-screw centrifuge tubes and domestic cryopreservation tubes, because these tubes are prone to tube rupture and sample loss when they are removed from liquid nitrogen. It is recommended to use the cryovials mentioned in Article 2. In the absence of conditions, the centrifuge tube must be fully sealed with parafilm to prevent explosion (this operation is not recommended)

3,Sample Shipping Precautions

 The upper limit of dry ice transport by aircraft is about 20 kg, and more than 20 kg cannot be transported in principle. Train transportation is not restricted.

② Dry ice transportation should use foam boxes with thick walls and good quality. Materials should be stored in numbered cryopreservation tubes, packed in plastic bags and then buried in dry ice. The foam boxes should be tightly buckled and sealed with sealing tape. Packed in a carton box to avoid cracking. And marked with light handling tips to ensure safe transportation

③ Dry ice transportation can be entrusted to a local express company, and try to ensure that it is delivered to the company within 48 hours.

④ For arrivals within 24 hours, the total amount of dry ice shall not be less than 5 kg; for arrivals within 48 hours, the total amount of dry ice shall not be less than 8 kg. In summer, you can add more dry ice (1.5 times the usual amount). Do not use bulk dry ice or dry ice that is completely powdered, but need small cylinders of dry ice, otherwise the bulk dry ice with a relatively large self-weight may squeeze the sample box in the box during transportation, which may cause the sample box to be moved. rupture. If only large pieces of dry ice can be used, break them into smaller pieces first when packing

⑤ When packing, it is recommended to wrap the dry ice in a large plastic bag and put an ice pack in the box, which can effectively slow down the evaporation rate of the dry ice.Use a foam box with a suitable size and thick wall, and wrap the transparent glue on the outside of the foam box in both vertical and horizontal directions. It is best to cover the outer wall of the foam box with tape to prevent it from being broken during transportation due to extrusion and throwing.After the foam box is all wrapped with tape, in order to prevent the seal from being too tight, the air tightness is too strong, and the volatilization of dry ice will generate excessive pressure and cause the foam box to burst,using a utility knife or other thin-edged knife, pierce a small gap of 1-2 cm with the tape wrapped around the junction of the foam box lid and the box to release the gas when the air pressure is too high.

⑥If you entrust express transportation, you should track the situation of the goods at any time during the transportation process, record the order number, and keep in touch with the courier company at the origin and destination.

4,Precautions

 If customers provide cryopreserved recoverable cell lines, they should first verify the reliability of the cryopreservation method used for RNA extraction, and provide detailed recovery methods when sending samples to ensure the success of the experiment,

② All samples should be marked with an accurate sample number, and a sample registration form should be provided, indicating the sample name, species, serial number, sampling date, and sample processing.

③ The quality and quantity of extracted RNA: a. The amount of RNA, RNA and protein contained in samples at different developmental stages and different growth conditions is different. The amount may be significantly different from the conventional corresponding samples; b. Storage conditions and storage time are also key factors affecting the quality and quantity of RNA. Generally speaking, the expected amount of nucleic acid can always be obtained from fresh samples, and the content is relatively high and easier to detect. However, samples that are not stored in liquid nitrogen or in a -80°C freezer are unreliable, so storage as low as possible is strongly recommended.

④ Regarding backup, in order to ensure the smoothness of the experiment, we strongly recommend that you back up 2-3 copies at the same time when sampling. If you back up 3 copies, send us two copies. If you back up 2 copies, send one sample. Keep a copy in case part of the sample degrades, re-collecting or sending samples will delay your time. Even if the samples are all qualified, you can use the backup samples for other experiments (such as quantitative verification, protein, biochemical experiments, etc.). There are two types of backups, one is strict backup, that is, after the sample is taken down, it is divided into two parts. Such two samples basically have the same nature. The other is a non-strict backup, that is, a biologically replicated sample. Such a backup sample is less homogeneous than the former.

5,Sample shipping guidelines

① Place the collected samples in the corresponding collection tubes. To prevent sample confusion, the cap and tube wall should be numbered and sealed with parafilm;

 Put the sample into a ziplock bag, fill in the sample details on the sample submission form (the information such as the serial number must be strictly consistent with the sample tube), and put the filled sample form and the sample into the foam box;

Use a foam box with a suitable size, a box wall greater than 4 cm and good sealing, and wrap the transparent glue on the outside of the foam box in both vertical and horizontal directions to prevent the foam box from cracking during transportation.

④ Sample delivery steps: