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mRNA integrity for qPCR

mRNA integrity qPCR

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#1 Dupasch

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Posted 09 June 2013 - 01:50 AM

Hey everyone,

I´ve tried to check my total RNA i isolated from whole tissue. Nanodrop ratio was ok. I´ve loaded the total RNA on a normal agarose gel (1%, 2ul Eth-Br, 130V, 30min). I saw the smear i didn´t whanted to see... within this smear two fade bands i think of to be the bands i was hoping for.

Now i have some quenstions:
  • I´ve already amplified a 500bp fragment from the cDNA made of this smeary RNA. The product sizes i whant to detect later in qPCR are 80-110bp. Is it possible that the RNA is partially degradadet but still could be used for qPCR?
  • is loading the cDNA made from RNA on a gel is leading to the same two bands?
  • Can i assume that if one sample from an extraction-charge is smeared, all other samples from this charge will be smeared too?

Thanks for every advice you can give me!!

#2 bob1

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Posted 09 June 2013 - 12:50 PM

Where was the smear on the gel - i.e. what sort of size was it? If it was smaller, say 50-500 bp ish then it may be degraded RNA, but if it is larger, around the 82s and 28s RNA bands then it is likely to be either that you have lots of RNA or some DNA contamination.

You can't assume between samples - you should run all samples to assess the integrity.

In theory the cDNA may give you those bands, but it would depend on how you prepared it and how much you have as to whether you would be able to see it on a gel.

#3 Dupasch

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Posted 09 June 2013 - 11:14 PM

Thats what it loked like. On the right the Ladder with bright bands at 5000 bp and 1500bp. from your desription i think it is not that much degraded??

smear.png

Edited by Dupasch, 09 June 2013 - 11:19 PM.


#4 bob1

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Posted 10 June 2013 - 01:07 AM

That is probably a high concentration of RNA species. Have a look around for sizes of the 28 and 18s RNA bands too.

#5 pmggms2

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Posted 11 June 2013 - 09:04 AM

So the proper ladder to use for RNA integrity is 50 bp and not 100 bp?

#6 Dupasch

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Posted 11 June 2013 - 11:19 PM

So the proper ladder to use for RNA integrity is 50 bp and not 100 bp?


The ladder i used had bright bands at 5000bp and 1500bp. a smaler ladder wouldn´t help you.

#7 Dupasch

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Posted 14 June 2013 - 05:12 AM

I did some samples with
a) normal gel
10062013.jpg

and b)denaturating Gel (MOPS)
14062013.jpg

The Samples are the same... in see small bands but in normal gel there is a smear in over these bands (that wouldn´t be a denaturation?) and the same samples on MOPS with smear under the two bands... i´m a little bit confused now... Could it be RNase in the chamber or smth like this? Are these probes worth trying qPCR??

Thanks again for any advice!





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