Aravind-

Trizol is great stuff and has worked for me with samples ranging from Archea to plants-- but it's not perfect and there are many possible reasons for it not working . Reading the original Chomzinski papers (where Trizol was developed) are very helpful. The patent is interesting reading too. Here are some ideas for troubleshooting your problem.

Biological:

Your sample may contain lots of dead or dying cells containing degraded RNA. Not much you can do about that if it is a property of your sample or a result of your treatment. Do a positive control extraction with cells you know are healthy to rule out methodological problems if you suspect this might be a biological problem.

Methodological:

Sample prep problems-

Flash freezing in liquid nitrogen is still the best IMHO. NEVER let the sample thaw before adding to Trizol. Work quickly- RNase released as a result of cell stress or loss of cell integrity can work amazingly fast.

Extraction problems-

1) By far, THE most common problem with Trizol is trying to extract too much tissue. Read the limits in the extraction protocol and follow them. Too much tissue and you overload the capacity of the chaotropic agents in Trizol and you get crappy RNA. More is not always better. When working with a new tissue, it is always good practice to first try a titration with different amounts of sample (again from cells you know are healthy). The amount of RNase in various samples varies wildly and what works for one sample may not work for another.

2) The second most common problem I've seen is getting greedy with the transfer of the aqueous phase after adding chloroform. Stay well away from the interface or you will get protein and gDNA carryover. I usually leave about 25% of the aqueous volume behind. btw- A trash spin and transfer to get rid of cell debris before adding the chloroform helps if you're getting a huge interface.

3) Other things I've seen as problems: Make sure the reagents used after you take off the aqueous phase are good quality RNase free reagents. Avoid damage from shearing-- e.g. If you are using bead-beating for cell disruption, keep that to a minimum. And avoid excessive pipetting up and down when redissolving pellets. Keep the samples at -80 as much as you can after extraction.

As general tips: It's always a good idea to include a positive control using healthy tissue that you know give good RNA. Doing pilot extractions with healthy tissue before committing your experimental samples can save a lot of headaches and a lot of time.

Good luck! Oh- and yes, the RIN needs to be as high as you can get it for RNA-SEQ. DEFINITELY over 7-- over 8 better.

The RIN value for my samples extracted 2 months back was as low as 2.6 and then it was immeediately rejected to send these samples for microarray. Now i have to do all the experiments once again and make sure that the RIN stays higher. I used trizol extraction method.

What could be the reasons for getting a low RIN......is it purely from experiment handling or any other reasons??

Cheers,

Aravind

There is a reason why you need high Quality RNA for most RNAseq protocols:

Before you fragment your RNA, you usually select polyadenylated transcripts through Poly(A) enrichment. Otherwise you would be sequencing all the ribosomal RNA (often 99% of your sample) and have no informative content in your library. However, if your transcripts are broken (= bad RNA Quality), you are mostly pulling out the ends of your transcripts near the polyA site and have no sequencing power (coverage) at the start of your transcripts. The result is a very uneven coverage across the length of your genes and little RNA input for the protocol in general.

I strongly advise you try everything to get your RIN score above 8. If you have no possibility to do so, your last resort is using a Protcol that does not rely on PolyA selection. One possibility is replacing the PolyA selection with RiboZero. Nevertheless, you will not get as good results as with a good RNA quality.

Hi Aravind,

RIN value as low as 2.6 is too low for any RNA experiments. Is not only experimental handling. Sometimes, when the sample was not properly stored, you can have RNA degradation and thus low RIN values.

Aravind-

Trizol is great stuff and has worked for me with samples ranging from Archea to plants-- but it's not perfect and there are many possible reasons for it not working . Reading the original Chomzinski papers (where Trizol was developed) are very helpful. The patent is interesting reading too. Here are some ideas for troubleshooting your problem.

Biological:

Your sample may contain lots of dead or dying cells containing degraded RNA. Not much you can do about that if it is a property of your sample or a result of your treatment. Do a positive control extraction with cells you know are healthy to rule out methodological problems if you suspect this might be a biological problem.

Methodological:

Sample prep problems-

Flash freezing in liquid nitrogen is still the best IMHO. NEVER let the sample thaw before adding to Trizol. Work quickly- RNase released as a result of cell stress or loss of cell integrity can work amazingly fast.

Extraction problems-

1) By far, THE most common problem with Trizol is trying to extract too much tissue. Read the limits in the extraction protocol and follow them. Too much tissue and you overload the capacity of the chaotropic agents in Trizol and you get crappy RNA. More is not always better. When working with a new tissue, it is always good practice to first try a titration with different amounts of sample (again from cells you know are healthy). The amount of RNase in various samples varies wildly and what works for one sample may not work for another.

2) The second most common problem I've seen is getting greedy with the transfer of the aqueous phase after adding chloroform. Stay well away from the interface or you will get protein and gDNA carryover. I usually leave about 25% of the aqueous volume behind. btw- A trash spin and transfer to get rid of cell debris before adding the chloroform helps if you're getting a huge interface.

3) Other things I've seen as problems: Make sure the reagents used after you take off the aqueous phase are good quality RNase free reagents. Avoid damage from shearing-- e.g. If you are using bead-beating for cell disruption, keep that to a minimum. And avoid excessive pipetting up and down when redissolving pellets. Keep the samples at -80 as much as you can after extraction.

As general tips: It's always a good idea to include a positive control using healthy tissue that you know give good RNA. Doing pilot extractions with healthy tissue before committing your experimental samples can save a lot of headaches and a lot of time.

Good luck! Oh- and yes, the RIN needs to be as high as you can get it for RNA-SEQ. DEFINITELY over 7-- over 8 better.

For sure RIN above 8 - close to 9 if you want to trust your results.

One of the biggest problems (i don't know what type of tissue you used) is also tissue storage prior to RNA extraction, and RNA storage itself - my experience is that proper storage is as relevant as RNA extraction methodology.

Degradation can also often be due to RNases.

I think you are mixing two different things: 1.to start with RNA of high quality RIN >7 which is very important since it tells you that you are not loosing a specific fraction of your RNA (the ones that can be easily degraded for example) with 2. Once you have the high quality RNA you fragment it. The thing is that you will fragment your RNA but all the expressed species will be represented.

This is completely different from starting with a bad quality RNA, where you cannot state that all RNAs are represented equally, and thus cannot come to conclusions regarding expression