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RNA prep help

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RNA prep help

Postby vastgenome » Mon Apr 05, 2010 2:31 am

Hi everyone,

I have been trying to prep RNA for qPCR and the yield has been extremely low even if i was very careful in handling things. I used QIAGEN's RNeasy Mini kit and the steps for RNA prep really is minimal. I followed the protocol to the letter, only taking liberty where it has been described as "discard the flow-through". the places where i think i may have messed things up are:
1. i let the lid of the column touch the liquid sometimes when i put them in a centrifuge
2. when discarding the flow-through, i decanted the liquid instead of pippetting it out. also i swung the tube a few times to get rid of any residual liquid as much as i can.
3. in the elution step i didn't let the water stand in column membrane for more than a minute, and directly centrifuged it into the collection tube.

i m really new in this business, so could anyone share some opinions? thanks!!!!!!!
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Postby JackBean » Mon Apr 05, 2010 6:04 pm

if you're using just water for elution, you should check pH of the water. Also, I think, that let it stand for at least a while is quite crucial (did you pipette it really into the middle?). The other thing, you can do two elutions (even with the same water). BTW what volume did you use?

Do you mean the system with the filters, right? So, it should be fine, if you just pour it out. But the crucial thing is, whether you remove all the ethanol?
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Re: RNA prep help

Postby domwood » Mon Apr 05, 2010 6:46 pm

I use RNAeasy Kits all the time and I find that the key factors are the quality of RNA and the control of contamination. Whatever you do to remove RNAses there will always be some present so I try to minimize the time between extracting RNA from it's native source and using it. If it's a tissue extraction I tend to get the tissue the same day as whatever you need the RNA for (qPCR/ cDNA etc.), if it's transcribed then the same, straight from the transcription reaction to RNAeasy to qPCR. Once you've got the reverse transcriptase in, you can relax so to speak.

If you can't use fresh RNA make sure it's stored in RNAlater or some other RNAse inhibiting solution. But in my experience the guanidine salts tend not to be all that good, also are you doing on-column DNAse digest, usually you can miss the step out.

Speed is the key thing for RNA. But other things I would suggest.

- You could try running the elutant a second time round (elute say in 30ul then take the 30 and run it through the column again)
- Treat whatever you can with DEPC.
- Anything you can't treat with DEPC, heat in an oven for 2 hours at 200'C then use it ASAP, RNAses are resistance to pretty much everything and after 2 hours out of the oven they begin to renature.
- I tend to have separate tips, separate glassware that I only use for RNA work and is labeled as such, also change your gloves regularly.
- Make sure you use nuclease free-water. use new water every time if you can.

Hope some of this may help.
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Re:

Postby vastgenome » Mon Apr 12, 2010 5:40 am

JackBean wrote:if you're using just water for elution, you should check pH of the water. Also, I think, that let it stand for at least a while is quite crucial (did you pipette it really into the middle?). The other thing, you can do two elutions (even with the same water). BTW what volume did you use?

Do you mean the system with the filters, right? So, it should be fine, if you just pour it out. But the crucial thing is, whether you remove all the ethanol?


thank you very much for the suggestions! the yield has been improved!

previously:
I used 14uL to elute RNA, although the kit recommends 20~30uL. for RNA stuff i always used barrier tips. I usually do the additional spin and collect the flow thru in a new 2mL tube. I usually use an RNase free cellstar cell culture lid to cover the column with the lid open and let the ethanol evaporate for about 1min. However, i never eluted the column more than once, nor did i let the water stand on membrane. i always added 14uL to the center and immediately take it to the centrifuge....i guess that ruined my yield...
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Re: RNA prep help

Postby vastgenome » Mon Apr 12, 2010 5:51 am

domwood wrote:I use RNAeasy Kits all the time and I find that the key factors are the quality of RNA and the control of contamination. Whatever you do to remove RNAses there will always be some present so I try to minimize the time between extracting RNA from it's native source and using it. If it's a tissue extraction I tend to get the tissue the same day as whatever you need the RNA for (qPCR/ cDNA etc.), if it's transcribed then the same, straight from the transcription reaction to RNAeasy to qPCR. Once you've got the reverse transcriptase in, you can relax so to speak.

If you can't use fresh RNA make sure it's stored in RNAlater or some other RNAse inhibiting solution. But in my experience the guanidine salts tend not to be all that good, also are you doing on-column DNAse digest, usually you can miss the step out.

Speed is the key thing for RNA. But other things I would suggest.

- You could try running the elutant a second time round (elute say in 30ul then take the 30 and run it through the column again)
- Treat whatever you can with DEPC.
- Anything you can't treat with DEPC, heat in an oven for 2 hours at 200'C then use it ASAP, RNAses are resistance to pretty much everything and after 2 hours out of the oven they begin to renature.
- I tend to have separate tips, separate glassware that I only use for RNA work and is labeled as such, also change your gloves regularly.
- Make sure you use nuclease free-water. use new water every time if you can.

Hope some of this may help.


Thank you very much domwood, I find your suggestions also very helpful, especially with the 2 rounds of elutions. i think you are absolutely right about the RNAlater one...the yield is always compromised with that solution. occasionally i would use liquid nitrogen to flash freeze my samples, but thawing and homogenizing the frozen samples often result in some kind of salt crystal that would go into the solvent, and i suspected that could have affected column binding conditions. now i use only fresh samples and the overall result is much better. i usually work w/ small sample sizes, ~900 cells, and for operational reasons some culture medium carryover is almost inevitable (~25uL aqueous solution) but indeed the fresh samples are superior than flash frozen ones, and far better than RNAlater.
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