a) Tissue Sample
1. Preparation: Freshly strip, clean, divide and store the tissues in TRIzol/RNAlater.
2. Dry Weight: >2g/sample. (Note: The DNA/RNA yields of different types of tissues vary greatly, and the specific sample delivery volume shall be subject to the quality inspection requirements.)
3. Means of Transport: Dry ice shipping within 72 hours.
4. Backup: In case of unqualified samples, customers are required to separately store the sample for backup before receipt.
5. Recommended processing method:
1) Pre-cool saline or PBS on ice (with the solution and utensils involved being DNase-free and Rnase-free);
2) Cut the desired tissue from the living body and place it on the plate; add pre-cooled saline or PBS to submerge the tissue, rinse to remove the blood stain, repeat the operations for 3 times or more (the time for ice operation should not exceed 10min) ;
3) Blot the surface droplets of the sample with a filter paper, cut the sample into small pieces of about 0.2-0.5cm (the smaller the tissue, the better the preservation effect);
4) Put the sample pieces into a centrifuge tube or cryotube with TRIzol or RNAlater;
5) Seal the frozen liquid nitrogen with a sealing film.

b) Cell Sample (including cultured microbial sample)
1. Preparation: Collect cells with high-speed centrifugation and store the cells in TRIzol/RNAlater.
2. Adherent or suspended cells: >5X106 cells/sample. (Note: The DNA/RNA yields of different types of cells vary greatly, and the specific sample delivery volume shall be subject to the quality inspection requirements.)
3. Microorganisms: Bacterial liquid in the logarithmic growth phase is recommended. Collect cells with high-speed centrifugation, store the cells into TRIzol/RNAlater after discarding the supernatant, and seal the frozen liquid nitrogen with a film.
4. Means of transport: Dry ice shipping within 72 hours.
4. Backup: In case of unqualified samples, customers are required to separately store the sample for backup before receipt.
6. Recommended processing method:
1) Pre-cool DNase-free and Rnase-free PBS on ice;
2) Adherent cells: Add a proper amount of pre-cooled PBS to rinse the cells after removing the medium, add 1mL of TRIzol to each 10cm2 of culture area, and gently blow all the cells for digestion and transfer to a centrifuge tube or a cryotube;
3) Suspended cells: Collect cells with centrifugation and remove the culture solution, rinse the cells with precooled PBS and add 1mL of TRIzol to each 5*106 cells, and gently blow all the cells for digestion and transfer to a centrifuge tube or a cryotube;
5) Seal the frozen liquid nitrogen with a sealing film.
The DNA/RNA sample extracted by customers should be subject to the quality inspection standard of RiboBio. Under normal circumstances, samples inspected by customers shall all be re-examined to ensure that the quality of the sample is not affected during transportation.

c) DNA Sample
1. Integrity: Genomic DNA requires gel electrophoresis to show that the main band is clearly distinguishable (mammalian genomic DNA main band >23Kb); PCR products are supposed to obtain a single band.
2. Purity: OD value at 1.6-2.0; no protein, RNA or visible contamination.
3. Concentration: >30ng/μl.
4. Backup: In case of unqualified samples, customers are required to separately store the sample for backup before receipt.
5. 1mL of solvent should be attached with the sample to dissolve DNA.
1) Pre-cool DNase-free and Rnase-free PBS on ice;
2) Adherent cells: Add a proper amount of pre-cooled PBS to rinse the cells after removing the medium, add 1mL of TRIzol to each 10cm2 of culture area, and gently blow all the cells for digestion and transfer to a centrifuge tube or a cryotube;
3) Suspended cells: Collect cells with centrifugation and remove the culture solution, rinse the cells with precooled PBS and add 1mL of TRIzol to each 5*106 cells, and gently blow all the cells for digestion and transfer to a centrifuge tube or a cryotube;
4) Seal the frozen liquid nitrogen with a sealing film.

Test Standards

1.The total amount of RNA (m) listed in the table above is mainly specified for human, mice and rats. For samples from other species, a higher m is recommended.

2.Microdatabase construction is different from conventional RNA-Seq in that it uses microlibrary construction kits with a relatively lower initial amount of the sample and a higher price. It is recommended only for samples with special sampling difficulties.

3.The small RNA library’s requirement on the total amount of RNA (m) is mainly specified for common cells, tissues and other samples. For special microRNA samples, such as serum, plasma, co-immunoprecipitation or exosome, m should be ≥200ng and c shoudle be≥1 ng/μL; for small RNAs (<200 nt) isolated by the customer, the sample volume should be ≥1 μg.

4.The concentration and total amount are based on the Agilent 2200 test results, and the purity is subject to the OD value of NanoDrop detection.

5.Level A samples are supposed to construct two or more databases, and Level B samples for one-time database construction. In order to ensure the smooth progress of projects, it is recommended to deliver samples according to Level A standards.

6.If the samples don’t meet any of the Level A or B standards and no spare samples can be supplied for special reasons, a risk database construction and sequencing can be conducted according to the actual situation, but the quality of sequencing is not guaranteed.

7.Should there be any problems, please consult local sales agents or technical support before sample delivery.

4) Requirements for RNA Samples

1. Completeness: 28S/18S>1.5; RIN>8.
2. Purity: OD value is between 1.8 and 2.0; no protein, RNA or visible contamination.
3. Concentration: >400ng/μl.
4. Backup: The customer is required to install the backup before receiving the sample, and analyze the reason when the sample is not qualified.
5. When transporting RNA samples, it is recommended to store in 70% ethanol (treatment method is as follows); if sending RNA after dissolution, please attach 1 mL of solvent used to dissolve RNA.

Purified RNA pre-treatment (recommended): After RNA quantification, adjust the volume to 100 or 200 μl, add 0.1 volume of 3M NaAc pH 5.2 (RNase-free) and 2.5 volumes of absolute ethanol; mix well below -20 °C The refrigerator was placed for more than 30 minutes; the RNA was precipitated by centrifugation at 13,000 g for 30 min at 4 ° C; the supernatant was discarded, 1.3 ml of absolute ethanol was added, and the ice was transported.

Detection Standard Specification

1.The total amount of RNA (m) listed in the table above is mainly specified for human, mice and rats. For samples from other species, a higher m is recommended.

2.Microdatabase construction is different from conventional RNA-Seq in that it uses microlibrary construction kits with a relatively lower initial amount of the sample and a higher price. It is recommended only for samples with special sampling difficulties.

3.The small RNA library’s requirement on the total amount of RNA (m) is mainly specified for common cells, tissues and other samples. For special microRNA samples, such as serum, plasma, co-immunoprecipitation or exosome, m should be ≥200ng and c shoudle be≥1 ng/μL; for small RNAs (<200 nt) isolated by the customer, the sample volume should be ≥1 μg.

4.The concentration and total amount are based on the Agilent 2200 test results, and the purity is subject to the OD value of NanoDrop detection.

5.Level A samples are supposed to construct two or more databases, and Level B samples for one-time database construction. In order to ensure the smooth progress of projects, it is recommended to deliver samples according to Level A standards.

6.If the samples don’t meet any of the Level A or B standards and no spare samples can be supplied for special reasons, a risk database construction and sequencing can be conducted according to the actual situation, but the quality of sequencing is not guaranteed.

7.Should there be any problems, please consult local sales agents or technical support before sample delivery.

Note：Sample Type

Level A samples refer to the samples qualified for database construction and the total amount is availale to build 2 or more databases.
Level B samples refer to the samples qualified for database construction and the total amount is only able to build 1 database.

Terms and Definitions

Qubit: Life technologies Qubit® 2.0 fluorescence, or DNA/RNA detection using Life technologies Qubit® 2.0 fluorescence.
Agilent 2100/2200: Agilent 2100/2200 Bioanalyzer, or DNA/RNA detection using the Agilent 2100/2200 Bioanalyzer
NanoDrop: Thermo Fisher NanoDrop ND-1000 or any micro-ultraviolet spectrophotometer, or a method for DNA/RNA detection using these instruments.
m: total mass (Total Mass), total amount of DNA/RNA.
c: Concentration, DNA/RNA concentration.
OD260/280: OD260/280 ratio, the ratio of 260 to 280 absorbance in DNA/RNA detection, reflecting DNA/RNA purity.
OD260/230: OD260/230 ratio, the ratio of 260 to 230 absorbance in DNA/RNA detection, reflecting DNA/RNA purity.