The key to successfully performing RNA-seq experiments is to provide the core facility with total RNA of sufficient quality and quantity to produce a library for sequencing. The quality of the initial samples is by far the single-most important factor in the whole process.
Tissue and Cell Isolation
Investigators need to carefully choose their methods of tissue and cell isolation, as these methods directly impact the quality and quantity of RNA that is subsequently obtained. If possible, total RNA purification should immediately follow tissue/cell isolation to prevent alterations in the transcript profile. However, in some cases, immediate RNA purification is not possible. If tissues/cells must be stored prior to RNA isolation then the use of products, such as RNALater from Qiagen or similar reagents, is recommended.
The core facility strongly encourages pilot projects to confirm that the chosen methods will reproducibly create sufficient quantities of cells/tissues to ultimately yield the required amount of high-quality RNA. Once an isolation protocol or a storage and isolation protocol is established, it is important that all of the samples collected for a given project be collected with this same protocol. Variance in these techniques may result in differences in the transcript profile. These differences may later be misidentified as changes due to experimental treatment, while in reality they are artifacts of varying isolation and storage methodology.
The facility does not perform total RNA purification. We require a minimum of 500 ng of total RNA for QC and library preparation for Illumina sequencing. A number of well-established commercial kits and protocols exist for a variety of species and tissue/cell types. Investigators carefully need to determine the most appropriate methods for their tissue/cell type. The core facility strongly encourages pilot projects to confirm that the chosen method will reproducibly create sufficient quantities of high-quality total RNA from the tissue/cell of interest. Given the tremendous variety of tissue/cell types, it is difficult for us to give specific recommendations. For anyone not sure of what product to choose, we strongly encourage you to examine the products from Qiagen and Ambion (LifeTech) as a starting point. These companies have a large variety of products with decision charts to help you in choosing the right one. In addition, if you need advice that is more detailed, there are people in tech support at these companies who truly are experts in RNA isolation and have a broad experience in helping customers isolate RNA from every conceivable species/tissue/cell. We however, do have a few general recommendations regarding RNA isolation techniques based upon our experience.
We do not recommend the use of Trizol alone for total RNA isolation, as the use of Trizol often results in samples that are contaminated by proteins and organics which can inhibit the library making process. We do recommend products like RNeasy, which is a column-based purification method producing very pure preparations of total RNA. Many of our customers have found though that they get superior yields with Trizol so they perform an initial isolation with Trizol followed by a further cleanup using the RNeasy kit. We have observed that this results in very pure RNA and this method has been used successfully for RealTime PCR, microarrays, and RNA-Seq.
Accurate Determination of RNA Concentration and Purity
RNA concentration is routinely determined by measuring absorbance at 260 nm. However, it should be noted that all nucleic acids have a peak absorbance at approximately 250 -260 nm; this includes RNA, DNA, and free nucleotides. If your RNA preparation contains contaminating DNA or free nucleotides it will affect your ability to determine accurately the RNA concentration in your sample with a spectrophotometer.
RNA purity is determined by measuring the 260/280 and 260/230 ratios using a spectrophotometer. Excessive absorbance at 280 indicates the presence of protein in your sample while excessive absorbance at 230 may indicate the presence of residual phenol in your sample. Ideally, the 260/280 ratio for RNA should be approximately 2.0 and the 260/230 ration should be 2.0-2.2.These ratios can be affected by pH so we are comfortable with all ratios over approximately 1.8. Ratios that differ significantly from that measure should undergo further purification.
We recommend the use of a NanoDrop to determine RNA purity and for an initial estimate of RNA concentration. This device is easy to use and less prone to error than traditional spectrophotometers because sample dilution is usually unnecessary and pipetting errors have no effect on correct determination of concentration. All facility customers may use the one we provide in 411 Chandlee, free of charge, but please bring your own pipettor and tips. This instrument is calibrated regularly so that customers may be assured it provides an accurate measurement.
We regularly see sample concentrations reported to us that are in wide variance from the true concentration measured by our facility with calibrated a NanoDrop or other techniques such as Bioanalzyer or Qubit. If you are using your own spectrophotometer please check that it is calibrated by measuring the concentration of commercially obtained standards, use calibrated pipettors, and be careful in calculating and performing your dilutions.
RNA Sample Quality
In addition to concentration and purity, it is essential to determine the quality of RNA samples prior to library preparation for RNA-Seq, to ensure that differential degradation of samples is not mistaken for differential expression. The quality of an RNA sample (its level of degradation) can’t be determined using the NanoDrop. Sample quality is determined using an Agilent Bioanalzyer. The Bioanalyzer will produce an RNA Integrity Number, or RIN, which is an objective measure of RNA quality. RIN scores vary from 1-10, with 10 being the highest quality samples showing the least degradation.
We not only like to see high RIN scores (7-10), but we also like to see a reasonably narrow range of the scores within a set of samples, which is typically 1-1.5. We recommend re-isolation of samples that have low RIN (6 or below) or are large outliers from the average RIN of a group of samples.
Ideally we would like 5 ul of total RNA at a concentration of 100 -200ng/ul to perform Bioanalzyer analysis and to confirm sample concentration and purity. Once the total RNA samples have passed these quality measures, the samples can be used for library preparation which requires approximately 300 ng of total RNA.
If the customer plans on performing rRNA depletion, we recommend checking each total RNA sample prior to depletion to confirm quality. Following depletion, the samples should once again be assessed on the Bioanalzyer to determine the success of the depletion.
- High sample quality is essential for successful RNA-Seq experiments.
- Customers are responsible for total RNA isolation.
- We encourage customers to perform pilot projects to determine the best tissue/cell isolation technique and RNA purification technique for their sample type.
- Once a tissue/cell isolation technique and RNA purification technique has been established it should be adhered to for all samples in a project.
- Determine total RNA sample purity and estimate sample concentration with a NanoDrop. The 260/280 and 260/230 ratios need to be greater than 1.8. Enough total RNA must be isolated to provide us with 500 ng for sequencing on the NextSeq or MiSeq.
- Determine total RNA quality prior to library construction by having the Genomics Core Facility assess the sample using the Agilent Bioanalzyer. RIN of 7-10 and ranges of RIN from 1-1.5 for a group of samples are preferred.