RNA-Seq Sample Recommendations
(Craig Praul, PSU and Caitlyn Bowman, Juniata)
The key to successfully performing RNA-seq experiments is to provide the core facility with mRNA 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 the choice of these
methods have a direct impact on the quality and quantity of RNA subsequently obtained. If possible
total RNA purification should follow tissue/cell dissection immediately to prevent alterations in the
transcript profile. 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 produce 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 quantitative differences in the transcript profile which may
later be misidentified as changes due to experimental treatment (for example, in RNAseq experiments)
while in reality they are artifacts of varying isolation and storage methodology.
RNA Isolation
The PSU facility does not perform total RNA purification nor does it perform rRNA depletion or mRNA
enrichment of total RNA. These processes are the sole responsibility of the customer. PSU requires a
minimum of 25 ng of mRNA or 200 ng of rRNA depleted RNA for library preparation for SOLiD
sequencing. The customer must isolate enough total RNA to ensure they will be able to produce
sufficient quantities of mRNA or rRNA depleted RNA. Typically mRNA may represent 1-5% of total RNA.
There are a number of well established commercial kits and protocols for a variety of species and
tissue/cell types. Investigators need to carefully 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 produce 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. Also if you need more detailed advice 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 do however 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
resulting in very pure preparations of total RNA. Many of our customers have found though that they
get superior yield 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 accurately determine 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 indicates the presence of protein in your sample while excessive absorbance at
230 may indicate the presence of residual phenol in your sample. The 260/280 ratio for RNA should be
approximately 2.0 and the 260/230 ratio 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 as 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 provided at the PSU genome
core facility in 411 Chandlee free of charge but please bring your own pipettor and tips. This instrument
is regularly calibrated 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 NanoDrops or other techniques such as
Bioanalzyer or Qubit discussed below. 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.
As noted above DNA may affect the accurate determination of RNA concentration. To measure the
presence of DNA in your sample we encourage the use of a Qubit which uses the specific binding of
fluorescently labeled dyes to determine DNA and RNA concentration. In our experience most RNA
preparations will contain some DNA contamination, often 5% or less. These molecules should not affect
the library construction process as they should not be amplified. The Qubit is a relatively inexpensive
device (the early model Qubit is only a few $100) and we recommend any labs routinely preparing
samples for Next-Gen sequencing to obtain one. If your does not have a Qubit we will check your
samples for DNA contamination as part of our QC checks prior to library preparation.
RNA Sample Quality
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 later mistaken for differential expression. 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. All total RNA samples should be run on the Bioanalzyer
prior to carrying out mRNA enrichment or rRNA depletion. We not only like to see high RIN scores (7-10)
but we like to see a reasonably narrow distribution of the scores 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. We require 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, rRNA depletion or mRNA enrichment
may be performed by the customer. Following depletion/enrichment the samples will once again be
assessed on the Bioanalzyer to determine the success of the depletion/enrichment.
.
Summary
1) High sample quality is essential for successful RNA-Seq experiments.
2) Customers are responsible for total RNA isolation as well as subsequent mRNA enrichment or
rRNA depletion.
3) We encourage customers to perform pilot projects to determine the best tissue/cell isolation
technique and RNA purification technique for their sample type.
4) Once a tissue/cell isolation technique and RNA purification technique has been established it
should be adhered to for all samples in a project.
5) 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
yield approximately 25 ng of mRNA or 200 ng of rRNA depleted RNA for sequencing on the
SOLiD.
6) Determine total RNA quality prior to mRNA enrichment or rRNA depletion by having the
Genomics Core 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.
7) Determine the success of mRNA enrichment or rRNA depletion by having the Genomics Core
assess the samples with the Agilent Bioanalyzer.