What are the steps for transcriptome sample extraction?

The core of transcriptomics sample extraction lies in the extraction and quality control of high-quality total RNA. The entire process must avoid RNA degradation and retain the original transcript expression information as much as possible. Here are the standardized steps:

 

I. Sample Preparation

• Sample types: Can be cells, tissues, fresh or frozen samples. It is recommended to immediately freeze with liquid nitrogen and store at −80°C or in RNA protective solution.

• Precautions: Avoid RNase contamination throughout, use DEPC-treated equipment and reagents.

 

II. RNA Extraction

Common methods include:

• TRIzol method (phenol/chloroform method): Suitable for most tissue samples, with high extraction efficiency;

• Column-based kits (such as Qiagen RNeasy): Simple operation, suitable for high-throughput sample processing, better purity but slightly lower yield.

Overview of steps:

1. Cell/tissue lysis: Add TRIzol or lysis solution, homogenize thoroughly.

2. Phase separation: Add chloroform, centrifuge to separate layers, collect the aqueous phase.

3. RNA precipitation: Add isopropanol, centrifuge to recover RNA.

4. Washing and resuspension: Wash with 70–75% ethanol, resuspend RNA in nuclease-free water.

5. (Optional) Removal of gDNA contamination: DNase I treatment (recommended as necessary).

 

III. RNA Quality Control

1. Concentration measurement: NanoDrop or Qubit, require A260/280 ≈ 2.0, A260/230 ≥ 1.8.

2. Integrity assessment: Use Agilent Bioanalyzer or TapeStation to measure RIN (RNA Integrity Number):

• RIN ≥ 7.0: Suitable for transcriptome library construction;

• If only used for 3' end sequencing, RIN ≥ 5 is acceptable;

• If the sample is severely degraded, consider using an rRNA removal scheme instead of polyA enrichment.

 

IV. Storage

• Short-term (<1 week): −80°C;

• Long-term: It is recommended to add RNA stabilizing solution (such as Ambion RNAstable) or store by lyophilization.

 

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