Many types of RNA-seq require RNA samples of high integrity and high chemical purity – please see the sample requirements. If the tissue or cell samples are handled correctly (e.g. flash frozen and stored at -80C) standard spin column RNA extraction kits will yield RNA samples perfectly suitable for RNA-seq. Please note that samples destined for miRNA or small RNA studies need to be isolated with protocols specifically designed to retain the small molecules (please see below). Standard RNA isolation protocols will lead to the loss and sequence-specific selection of small RNA molecules. RNA samples should always be DNA-free. Nanodrop readings are more or less useless to determine RNA sample concentrations – please use fluorometric quantification instead (e.g. Qubit or Quantus instruments). The Nanodrop readings should be used to assess sample purity.
- For most tissues, the standard Qiagen RNeasy kits (cat. no. 74004) are perfectly fine (or similar kits from other vendors). These RNAeasy kits have to be used in conjunction with the Qiagen RNase-Free DNase kit (cat. no. 79254). We recommend to isolate the RNA, then perform the DNAse digestion in solution on the isolated RNA, and then clean up this reaction once more with the RNeasy kit. Alternatively, a kit like the Qiagen RNeasy Plus Micro Kit (cat. no. 74034) with “DNA Eliminator” gDNA removal columns may be used.
- In case the samples contain interfering chemicals or in case of very small sample amounts, it could be worth trying kits from Norgen Biotek. This manufacturer offers a selection of sample-type specific kits and uses a proprietary silicon carbide spin column matrix, which has a higher affinity for RNA compared to the standard silica columns. Thus, the Norgen Biotek kits often provide higher yields. Norgen kits offer two options for DNA removal: “plus” kits come with a dedicated DNA removal column; standard kits OTOH require DNAse treatment with the DNAse digestion add-on (Norgen cat. no. 25710).
- Always perform at least two spin column washes (with the kit wash buffer) after binding of the lysed sample to the column matrix.
- In case your lab uses a Trizol protocol for RNA isolations, we do recommend an additional sample purification with a spin column kit (e.g. Zymo RNA clean & concentrator 5 kit with DNAse – cat. no. R1013) for a DNAse digestion and clean up.
- An additional column cleanup is mandatory for RNA samples isolated from blood PAXgene or Tempus tubes (the tubes are used for blood preservation) after the initial RNA isolation. Often the preservative chemicals tend to contaminate the samples upon isolation. For the cleanup, we recommend the Zymo RNA clean & concentrator 5 kits with DNAse (cat. no. R1013) or similar.
- For miRNA and small RNA studies, protocols specifically designed to isolate also the shorter molecules have to be employed. Appropriate kits are available from multiple suppliers. Some suitable examples are an array of sample-type-specific NorgenBiotek and Qiagen kits as well as the Zymo Quick RNA kit. Please make sure that you apply the protocol variants designed to retain miRNAs for all of these.
- NEVER use heparin as an anticoagulant for blood samples destined for DNA or RNA sequencing.
- Avoid using glycogen (often recommended as co-precipitant).
- The 260/230 nm and 260/280 nm absorption ratio measurements (e.g. from NanoDrop) are should be used to assess sample purity. For RNA the 260/230nm ratio should be >1.5 and the 260/280nm ratio 1.8-2.1; Please see the sample requirements page and the sample cleanup FAQ.
How many cells will be needed to isolate sufficient RNA for conventional RNA-seq?
The typical mammalian cell contains 10 to 30 pg of RNA. Assuming the worst-case scenario (only 10 pg RNA content; 50% loss during isolation), you should be starting the RNA isolation with 20,000 or more cells to reach at least 100 ng total RNA sample, the lowest amount recommended for RNA-seq after poly-A enrichment. Please see the small sample RNA isolation recommendation above.
Avoiding Batch-Effects:
Both sample storage conditions and details of the RNA-isolation protocols are well-known to introduce technical variations into RNA-seq data. Because of this, it is recommended to:
- Isolate the RNA-samples in one batch.
- If RNA-isolations need to be carried out in several batches, they should be carried out by the same person using the same batch of reagents
- If RNA-isolations need to be carried out in several batches, the samples should be randomized between the RNA isolation batches (worth discussing with a statistician or the Bioinformatics Core).
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