RNA half life issue for transcriptome sequencing

[harshinamdar]

I have a very basic question. RNA has a very short half life, how is it handled during RNA-seq experiments?
Does it not get degraded before being read by sequencer?

[steven]

Considering the amount of data generated, it looks like not all of the RNA is degraded before being actually sequenced
Usually there is a RNA-to-cDNA (Reverse Transcription) step going on, but it looks like even direct RNA sequencing produces some consistent signal.
So: short half life indeed, but not too short.

[dariober]

Consider also that RNA is degraded by RNAse's. If you work in RNAse free conditions the half life of the RNA should be long since chemically it is quite stable.

[harshinamdar]

Thank you steven and dariober . that helps a lot.
Another question ,if i can ask , is the time period for which the RNA sample is collected?
is there a fixed time limit to get a consistent sample of RNA?

[steven]

You could start by having a look at this paper maybe.

[shurjo]

Thank you steven and dariober . that helps a lot.
Another question ,if i can ask , is the time period for which the RNA sample is collected?
is there a fixed time limit to get a consistent sample of RNA?

If I understand correctly, are you referring to the post-mortem interval for RNA extraction from tissue? If so, in my samples this can be as long as 2-7 hours before the RNA shows substantial degradation, assuming that the ambient temperatures for the cadaver was ~70 degrees F.

[malachig]

How to avoid RNA degradation

A few general comments. The RNA in a live cell exists in a state of equilibrium between synthesis and degradation. This is why RNA-seq and microarray experiments are commonly referred to as a 'snapshot' of steady state transcription. As mentioned by dariober, the primary cause of RNA degradation is the action of RNases. Once cells are removed from an active state of maintenance (e.g. removal of media in culture, removal of a tumor from a patient, death of the organism), the balance will shift towards degradation.

There are several ways to prevent this: (1) Temperature. As mentioned by shurjo, RNAse activity is inhibited at very low temperatures. If you can not process cells or tissue immediately then they should be frozen to -75C as quickly as possible. Many tumor tissue banks have protocols that require the sample to be flash frozen within 30 minutes to minimize RNA degradation. (2) Chemical inhibition. One common strategy is to homogenize the tissue/cells immediately and place in a lysis buffer that creates an environment that prevents RNAse activity, even at room temperature. For example, the widely used Trizol (modified phenol:chloroform extraction) and column based total-RNA isolation systems include a chaotropic agent such as Guanidinium thiocyanate. These agents denature proteins and prevent their activity until they can be removed. The same principle is possibly used by RNA/Tissue preservatives such as 'RNAlater', something to consider if you need to store tissue before isolating RNA but do not want to freeze it. (3) Use of protein based RNAse inhibitors. If your RNA isolation requires an initial step in buffer that does not contain a 'harsh' buffer containing chaotropic agents (such as during cytoplasmic RNA isolation protocols), you may want to consider addition of other RNAse inhibitors such as SUPERaseIn. (4) Protein precipitation. In combination with the steps above, the best outcome is to remove all proteins (including RNAses) from your RNA as soon as possible. The RNA can then be stored indefinitely in a freezer and used in downstream applications at room temp. without degradation. Most total RNA isolation kits are highly effective at removing protein content. (5) Use of 'proper' RNA handling methods. Even if you are successful in protecting your RNA from endogenous RNAses (i.e. those in the original cells), you still need to avoid exposure to outside RNAses. These could be RNAses used for other protocols in your labs (common) or from your hands, etc. Many library construction groups thus create an 'RNA only' area where RNAses from other protocols are forbidden, anti-RNAse cleaning agents are used (e.g. 'RNAse zap'), gloves are always worn, etc. (6) Conversion to cDNA. As mentioned by steven, cDNA synthesis from RNA using a reverse transcriptase is a common step in library construction. The resulting cDNA is more stable than RNA ... although it then becomes subject to degradation from DNAses.

Your question about storage time is important. As discussed above, the length of time as well as the manner in which your tissue/cells are stored may have a significant impact on RNA degradation levels. In my experience the highest quality RNA is most easily obtained from cells grown in culture. One minute they are healthy and growing and the next minute they are immersed in a buffer with RNA protection properties (with the intervening time of ice). We have had good results taking cultures from an incubator, rinsing with ice cold buffer (PBS for example), and then adding Trizol directly to the plate and gathering and homogenizing the cells by pipetting. We have also obtained reasonably high quality RNA from fresh frozen tumors from a tumor bank using standardized collection and storage procedures. With FFPE tissues that have been stored at room temp for extended periods, no isolation procedure or practice will yield high quality RNA (the damage has already been done)...

If you provide more details of the source of your RNA (species?, tissue?, storage method?, cultured?, resected?, post mortem?, etc.) someone with directly relevant experience may be able to provide more specific guidance.

[malachig]

Assessing quality of RNA

To determine how well your tissue handling, storage and RNA isolation procedures are working, it is important to accurately and consistently assess the quality and quantity of total RNA obtained before using it in downstream applications (particularly expensive and time consuming experiments such as RNA-seq). In our group we use both spectrophotometry and capillary electrophoresis for this purpose.

A 'NanoDrop' or other spectrophotometer allows you to quantify RNA solutions and detect the presence of contaminants such as protein, salt, guanidinium thiocyanate, ethanol, phenol/chloroform, etc. that may have made it through the isolation procedure. These contaminants can interfere with downstream applications (particularly enzymatic reactions) so their presence may cause you to repeat the isolation or perform further clean-up reactions. A good total RNA solution should have high 260/280 and 260/230 ratios (>=2 if possible).

We use an Agilent 2100 Bioanalyzer (RNA 6000 Nano assay) for capillary electrophoresis assessment of RNA degradation. These assays replace the more traditional approach of running RNA on a gel, visualizing, and qualitatively assessing RNA integrity. Instead you get a quantitative estimate of RNA yield and both a qualitative and quantitative assessment of RNA integrity. The qualitative information comes in the form of an electropherogram (peaks corresponding to relative amounts of RNAs by size). The quantitative information comes in the form of an RNA Integrity Number (RIN) (a score from 0-10). Various library construction groups have determined the minimum RIN that tends to yield a successful RNA-seq library (our group prefers 8 or higher). The topic of RIN has been specifically discussed in this forum here: Total RNA quality (Bioanalyzer trace attached)

[steven]

Thanks a lot Malachi, that is great!

[harshinamdar]

Thanks a ton everyone.