Getting Started with RNA Sequencing
RNA-seq (RNA sequencing) has undoubtedly become the most popular method for transcriptome analysis. It is widely used for gene expression analysis, including detection of mutations, fusion transcripts, alternative splicing, and post-transcriptional modifications.
Creating a RNA-Seq Library
Standard methods for RNA library preparation do not retain information on the DNA strand from which the RNA strand was transcribed. The ability to obtain information on the originating strand is useful for many reasons including the identification of antisense transcripts, determination of the transcribed strand of noncoding RNAs, and determination of expression levels of coding or non-coding overlapping transcripts. Overall, the ability to determine the originating strand can substantially enhance the value of a RNA-seq experiment.
Even beyond the decision to perform direction or non-directional library prep, several methods can be used to generate an RNA-seq library, and the details of these methods are dependent on the platform used for high-throughput sequencing. However, there are common steps.
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- Abundant Transcript Removal: The majority of RNA molecules present in a cell are ribosomal RNA (rRNA), and since these are generally not of interest, they should be removed before making a library from the RNA of interest. Similarly, globin RNA is generally removed from blood samples and chloroplast RNA is often removed from plant leaf samples. Two popular options for this step are:
- mRNA isolation: This involves targeting the polyadenylated (poly(A)) tails to ensure that non-coding RNA, including rRNA, is separated from polyadenylated transcripts.
- rRNA depletion: Total RNA can be depleted of rRNA using a number of methods, most of which involve hybridization of oligos to the rRNA followed by removal. rRNA depletion enables subsequent sequencing of all non-rRNA molecules, and is not limited to intact mRNA molecules.
- Fragmentation: Fragments of an appropriate size for sequencing are generated by fragmentation of RNA prior to reverse transcription and cDNA synthesis, rather than by fragmentation of cDNA.
- Reverse transcription and second-strand cDNA synthesis: Complementary DNA (cDNA) is generated from the RNA template by a reverse transcriptase. This first strand cDNA is then made double stranded using a DNA polymerase.
- End repair, dA-Tailing and Adaptor Ligation: End repair of the ds cDNA library and optional dA-tailing (depending on the sequencing platform to be used) is followed by ligation to adaptors. The library is then ready for amplification and sequencing.
Important factors to consider when performing RNA-seq
Library preparation is an important part of the RNA-seq workflow and methods are currently available for library preparation for RNA-seq which offer simplified protocols and improved yields. However, the quality and accurate quantitation of input RNA still remains critical to ensuring successful cDNA synthesis and libraries. The following are some important factors to consider:
RNA Sample Input Guidelines
Integrity of RNA:
Quantitation of RNA
- It is important to start with high quality RNA. The use of degraded RNA can result in low yield or failure to generate libraries. We recommend determining RNA quality using the RNA Integrity Number (RIN) estimated by the Agilent® Bioanalyzer®. The RNA sample should have a RIN value higher than 7.
- The integrity and size distribution of total RNA can be checked by electrophoresis on a denaturing agarose gel and staining with ethidium bromide. The ribosomal RNA bands should appear as sharp bands on the stained gel. For eukaryotic samples, intact total RNA will have sharp, clear bands corresponding to 28S and 18S. The 28S rRNA band should be approximately twice as intense as the 18S rRNA band. This 2:1 ratio (28S:18S) is a good indication that the RNA is completely intact. Partially degraded RNA will have a smeared appearance, will lack the sharp rRNA bands, or will not exhibit the 2:1 ratio of high quality RNA. Completely degraded RNA will appear as a very low molecular weight smear.
- RNA should be completely free of DNA. DNase digestion of the purified RNA with RNase-free DNase is recommended.
- It is important to quantify accurately the RNA sample prior to library construction. The concentration can be estimated with the Agilent Bioanalyzer on a pico or nano chip. Alternatively, RNA concentration can be determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer such as a NanoDrop®. However, free nucleotides or other organic compounds routinely used to extract RNA will also absorb UV light near 260 nm and will result in an overestimation of the RNA concentration.
View this video for additional tips on optimizing inputs for RNA sample prep.