What is inhibiting my RTPCR? - Are environmental samples too complex? (Sep/16/2007 )
Hello,
I am checking the copy numbers of two different microbial genes in the environment. I have isolated RNA from environmental samples, reverse-transcribed from the RNA, and then made a dilution series (6 serial 2X dilutions, to 1/32) of the RT product. I use QPCR on each of the dilutions.
When I use this approach with my controls (pure cultures - ie, single species of bacteria that we grow) I see the expected result:
1X dilution: 5960 copies
2x dilution: 2830 copies
4X dilution: 1750 copies
8X dilution: 800 copies
16X dilution: 315 copies
32X dilution: 140 copies
But, when I use this approach with my environmental sample, the numbers are not linear at all (although there is a decrease, once you get past the undiluted sample):
1X dilution: 85 copies
2x dilution: 569 copies
4X dilution: 494 copies
8X dilution: 396 copies
16X dilution: 262 copies
32X dilution: 213 copies
These patterns are reproducible - the cultures always give the expected result and the environmental samples don't. There is obviously some sort of inhibitor in the environmental sample, and I would like some help figuring out what it is/how to get around it.
I use RNeasy to extract RNA from both the cultures and the environmental sample (mud). I use the High Capacity RT kit to make cDNA. Is there any "old school" cleanup method that might get rid of the inhibitor (like cesium banding or something)?
Is it possible that an environmental sample has so much more complexity (in terms of cDNA species) than a pure culture, that the target is harder to reliably amplify in QPCR? (Could a complex mix of cDNA fragments be the inhibiting compound)?
Is it possible that an environmental sample has so much more complexity (in terms of cDNA species) than a pure culture, that the target is harder to reliably amplify in QPCR? (Could a complex mix of cDNA fragments be the inhibiting compound)?
OK. sounds... dirty. LOL. hopefully my comments will help.
First off, I would say that the Qiagen RNAeasy kit was not designed to extract RNA from MUD. You might want to try a kit from OMEGA Bio-Tek that was designed specifically for this (its a small company we used to get stuff from when I was in grad school), here's a link
E.Z.N.A.™ Soil RNA Isolation Kit
http://www.omegabiotek.com/productsrange/R...ll_rna_kit.html
But... regardless; once you have a purifed RNA sample what is the OD260/280? and the OD260/230? These values will give you some idea as to the quality (saltiness) and purity (absence of proteins). But i assume you already know that - just saying so for coverage. If your purity and quality ratios are good, my next question would be - what's the concetration of your sample? If it is dilute to begin with (<10 ng / ul ) then I would recommend concentrating it using a simple ethanol precipitation @ -80oC. however... if you have good concentration, quality and purity, then it's probably more to do with your environmental sample itself. So, try doing qPCR on a positive control gene that is unique to your target organism AND a second positive control that is common to most everything (try 18S rRNA, all life on earth has that one). Hope that helps. let us know if your situation improves and what did the trick.
GOOD LUCK!
Is it possible that an environmental sample has so much more complexity (in terms of cDNA species) than a pure culture, that the target is harder to reliably amplify in QPCR? (Could a complex mix of cDNA fragments be the inhibiting compound)?
OK. sounds... dirty. LOL. hopefully my comments will help.
First off, I would say that the Qiagen RNAeasy kit was not designed to extract RNA from MUD. You might want to try a kit from OMEGA Bio-Tek that was designed specifically for this (its a small company we used to get stuff from when I was in grad school), here's a link
E.Z.N.A.™ Soil RNA Isolation Kit
http://www.omegabiotek.com/productsrange/R...ll_rna_kit.html
But... regardless; once you have a purifed RNA sample what is the OD260/280? and the OD260/230? These values will give you some idea as to the quality (saltiness) and purity (absence of proteins). But i assume you already know that - just saying so for coverage. If your purity and quality ratios are good, my next question would be - what's the concetration of your sample? If it is dilute to begin with (<10 ng / ul ) then I would recommend concentrating it using a simple ethanol precipitation @ -80oC. however... if you have good concentration, quality and purity, then it's probably more to do with your environmental sample itself. So, try doing qPCR on a positive control gene that is unique to your target organism AND a second positive control that is common to most everything (try 18S rRNA, all life on earth has that one). Hope that helps. let us know if your situation improves and what did the trick.
GOOD LUCK!
Thank you Jonathan,
Today I decided to try simply running the RNA and RT products over a size exclusion column. THe RNA seemed to benefit from it - it gave a better absorption profile afterwards. I will perform the QPCR tomorrow, and will let you know if the cleanup helped.
I've made a note of the Omega bio tek product, thank you!
So now I am running the RNA through a cesium gradient, because I still have an inhibiting compound. I made a lysate of my environmental sample, and layered it on a 4.7 molar cesium cushion. I am spionning overnight. The inhibitors, in theory, will sit on top of the cesium cushion. I plan to take the RNA pellet, and bind it to a column in order to further clean it and elute it in a small volume.
Here is my next question:
Will the cesium chloride in the RNA pellet (4.7M) prohibit binding between my RNA and the RNAeasy spin column? Do I need to somehow remove the CsCl first?
Try using part of your sample (for your cDNA synthesis) and do multiple 70% ethanol precipitations @ -80oC to "wash out" the salts. Do the washes at least three times. Each wash you may loose a little RNA, but you will be greatly reducing the CsCl in your sample. Once you are done with this, you may not even need to use your RNAeasy columns.
Check out this protocol for some more information
http://crawford.rsmas.miami.edu/Protocols/...%20Chloride.doc
LET US KNOW HOW IT GOES!
I thought about precipitating with ethanol, but I need the final prep to be free of DNA, and the RNAeasy columns are supposed to preferentially bind RNA over DNA. I may need to go back and do it the way you suggest, though. If so, I will need to add a DNAse step.
I was very impressed with the ultra-spin..... There was a green slime on top of the cesium cushion, and more pigmented junk in the upper lysate layer.
The lower cesium layer looked fairly clean. There were some particulates that pelleted, and the RNA pelleted (as expected.)
I re-suspended the pellets in TE and spun out the particulates, and then processed the TE/RNA on an RNeasy column. It appears that any cesium carryover did not inhibit binding to the column.
At the ethanol step, the RNA was white and fluffy, instead of questionable-looking as in past preps. I do not know if any cesium carried over to the final eluate, off the column. My final concentration was 123 ng/microliter which is low but plenty. The UV absorption profile was very good. I have RT'd the sample and wil QPCR in the next day or two.
The ultra spin was very easy. If it is this simple to clean up RNA from mud, I'm all for it.
I was very impressed with the ultra-spin..... There was a green slime on top of the cesium cushion, and more pigmented junk in the upper lysate layer.
The lower cesium layer looked fairly clean. There were some particulates that pelleted, and the RNA pelleted (as expected.)
I re-suspended the pellets in TE and spun out the particulates, and then processed the TE/RNA on an RNeasy column. It appears that any cesium carryover did not inhibit binding to the column.
At the ethanol step, the RNA was white and fluffy, instead of questionable-looking as in past preps. I do not know if any cesium carried over to the final eluate, off the column. My final concentration was 123 ng/microliter which is low but plenty. The UV absorption profile was very good. I have RT'd the sample and wil QPCR in the next day or two.
The ultra spin was very easy. If it is this simple to clean up RNA from mud, I'm all for it.
The cleaned RNA had significant DNA contamination. So, I added a DNAse step and purified the RNA over a column, again.
At this point the RNA is beautiful, with no DNA contamination. The RT product behaves very nicely.
So, the upshot of all this is that the inhibitor from environmental samples can be removed by running the initial lysate through an overnight ultraspin on a cesium cushion (4.7M). The resulting nucleic acid needs to be DNAsed, but can be purified on RNeasy columns in good quality.
Possibly an ethanol precipitation would also do the trick, but for the time being I have a simple method that seems to work, so I'll stick with it. Thank you for your help, Jonathan.