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Reverse-transcriptase PCR troubleshoot HELP - (Jun/23/2005 )

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Using Invitrogen's Superscript III First strand synthesis Supermix and followed by Accuprime Supermix PFX for cDNA amplification.

I've been trying to get my RT-PCR to work but I keep on having very very little to no bands when I run the cDNA product on an agarose gel. My starting RNA is harvested from mouse liver using the Qiagen kit and I used oligo dts. Not even the + control HeLa included in the superscript kit is giving me a strong band. I started using Extaq from Takara with no success so I switched to Accuprime Supermix but still no good. I checked the cDNA primers with a mouse liver panel as a positive control and they came out great. What am i doing wrong?

-k81878-

how much time has passed since your RNA extraction and cDNA synthesis?
RNA doesn't last very long, and should be used as soon as possible,and should be kep at -80C.
other than that, maybe you could fiddle around with the temperatures on your PCR.
Vetticus

-vetticus3-

Try random hexamers instead of oligo dt. I have a terrible time getting good pcr off of cDNa with oligo dt and have excellent results with hexamers.

Promega makes a good one, dilute 1:10 with depc water and use 2ul in a 20ul rxn.

-pBluescript-

How much starting RNA are you using in your RT step?

When you say cDNA product, are you talking about the cDNA product produced by your RT step or your PCR product.

You are not going to produce a lot of cDNA using dt oligos since they target mRNA which only makes up 1-3% of total RNA per cell. So looking at it on an agarose gels won't help much unless your starting RNA is more than a couple of ugs.

If your PCR is weak it could mean three things. You primer targets are too far from the polyA tail of targeted mRNA and you need to switch to hexamer primers like the gentleman earlier stated.

or..

your target mRNA copy number per cell is small and you need to increase your starting amount of RNA in your RT step to produce reliable results.

or..

one of the other few dozen of things that can go wrong with RT-PCR even though you did exactly what the protocol said.

-dobbiewalton-

QUOTE (dobbiewalton @ Jun 24 2005, 12:13 PM)
How much starting RNA are you using in your RT step?

When you say cDNA product, are you talking about the cDNA product produced by your RT step or your PCR product.

You are not going to produce a lot of cDNA using dt oligos since they target mRNA which only makes up 1-3% of total RNA per cell.  So looking at it on an agarose gels won't help much unless your starting RNA is more than a couple of ugs.

If your PCR is weak it could mean three things.  You primer targets are too far from the polyA tail of targeted mRNA and you need to switch to hexamer primers like the gentleman earlier stated.

or..

your target mRNA copy number per cell is small and you need to increase your starting amount of RNA in your RT step to produce reliable results.

or..

one of the other few dozen of things that can go wrong with RT-PCR even though you did exactly what the protocol said.


My boss doesn't want me to use random hexamers yet....

I meant the cDNA product from after pcr amplification of target cDNA. But now that you mentioned it, what if I run on a gel a big batch of first strand cdna product from RT-PCR (like 200-400 ul?). And then do gel extraction? Is that possible? If it is, should I treat it as an RNA and be worried abt contamination when loading it on an agarose gel?

-k81878-

[/quote]

My boss doesn't want me to use random hexamers yet....

I meant the cDNA product from after pcr amplification of target cDNA. But now that you mentioned it, what if I run on a gel a big batch of first strand cdna product from RT-PCR (like 200-400 ul?). And then do gel extraction? Is that possible? If it is, should I treat it as an RNA and be worried abt contamination when loading it on an agarose gel?


[/quote]

I wouldn't run a gel on it. You won't get as a discrete band on cDNA as you would on PCR product. RT is affected by the varying complexity of the mRNA sequences. So the cDNA will be of varying lengths.

You need a way to check your RT reaction. Did you spec your RNA before the RT reaction? Was it of good quality? How about instead of running 200-400 ul of cDNA on a gel, you check 5-10 ul on a spec. If you have treated the RT-reaction with RNaseH, then you can look at the amount of cDNA you have produce. If the amount of cDNA is close to the amount of starting RNA, your RT reaction is fine. If not, you need to repeat.

-dobbiewalton-

QUOTE (dobbiewalton @ Jun 28 2005, 11:20 AM)
I wouldn't run a gel on it.  You won't get as a discrete band on cDNA as you would on PCR product.  RT is affected by the varying complexity of the mRNA sequences.  So the cDNA will be of varying lengths. 

You need a way to check your RT reaction.  Did you spec your RNA before the RT reaction?  Was it of good quality?  How about instead of running 200-400 ul of cDNA on a gel, you check 5-10 ul on a spec.  If you have treated the RT-reaction with RNaseH, then you can look at the amount of cDNA you have produce.  If the amount of cDNA is close to the amount of starting RNA, your RT reaction is fine.  If not, you need to repeat.



So if the amt of cDNA is more than the starting RNA it's bad?


When I first tried to spec the total RNA sample that I got (somebody else harvested it using the qiagen kit) the spec can't even read anything so I switched to Invitrogen's dipstick and got an estimate of abt 1-2 ng/ul. Before amplifying the target cDNA, I also spec and I got abt 1.2 ug/ul. I tried doing a template gradient using a diluted template of abt 200 ng to 4 ug of synthesized 1st strand cdna but the results are inconsistent. Even my positive control (HeLa) is weird. Sometimes the band would show but most times it won't or very very faint. =(

Is there a way I can purify the Total RNA sample (kept in the -80 freezer)that I have and concentrate it? Like get rid of all the other rna and just get mRNA?

THANKS VERY MUCH for all the help wacko.gif

-k81878-

QUOTE (k81878 @ Jun 30 2005, 06:02 PM)
QUOTE (dobbiewalton @ Jun 28 2005, 11:20 AM)


I wouldn't run a gel on it.  You won't get as a discrete band on cDNA as you would on PCR product.  RT is affected by the varying complexity of the mRNA sequences.  So the cDNA will be of varying lengths. 

You need a way to check your RT reaction.  Did you spec your RNA before the RT reaction?  Was it of good quality?  How about instead of running 200-400 ul of cDNA on a gel, you check 5-10 ul on a spec.  If you have treated the RT-reaction with RNaseH, then you can look at the amount of cDNA you have produce.  If the amount of cDNA is close to the amount of starting RNA, your RT reaction is fine.  If not, you need to repeat.



So if the amt of cDNA is more than the starting RNA it's bad?


When I first tried to spec the total RNA sample that I got (somebody else harvested it using the qiagen kit) the spec can't even read anything so I switched to Invitrogen's dipstick and got an estimate of abt 1-2 ng/ul. Before amplifying the target cDNA, I also spec and I got abt 1.2 ug/ul. I tried doing a template gradient using a diluted template of abt 200 ng to 4 ug of synthesized 1st strand cdna but the results are inconsistent. Even my positive control (HeLa) is weird. Sometimes the band would show but most times it won't or very very faint. =(

Is there a way I can purify the Total RNA sample (kept in the -80 freezer)that I have and concentrate it? Like get rid of all the other rna and just get mRNA?

THANKS VERY MUCH for all the help wacko.gif




I hope that was a misprint. Getting more cDNA than intial amount of RNA is normally unlikely since the cDNA being produced forms a RNA/DNA hybrid with the RNA. Remember this a transcription not an amplification. So its highly unlikely you got 1 ug/ul of cDNA from 1ng/ul of RNA.

If that was a misprint, did you treat you cDNA to get rid of the RNA? How much of the cDNA are you using for your PCR reaction? Try using different amount of cDNA. If your reaction is 20ul try using 5 ul of cDNA, 5ul of 1:2 dilution, 5ul of 1:5 dilution and 5ul of 1:10 dilution. Too much cDNA can cause inhibition of your PCR. The SuperScript II kit usually recommends 50ng to 5 mg of RNA. I typically use anywhere between 500ng-1ug of intial RNA. I then use 5ul of cDNA with no problems. But sometimes I do a dilution with difficult mRNA targets and it helps in 25% of the cases.

It also important to use everything RNase free and/or depc treated. I remember doing a RT-QPCR a while back and i couldn't get anything. I traced it back to some non-nuclease free 15ml conical tubes i used during RNA extraction.

I not sure if there is a way to isolate mRNA from a total RNA extraction.

-dobbiewalton-

Sure there is a way to get mRNA from total RNA.. oligo dT cellulose.

-pBluescript-

QUOTE (pBluescript @ Jul 1 2005, 12:48 PM)
Sure there is a way to get mRNA from total RNA.. oligo dT cellulose.



so..... is that a good idea? By now it's pretty obvious that I don't really know my way around RT-PCR. =P So if i do an Oligo dt cellulose step using the total rna that I have before going for RT-PCR, does that sound like a good plan?

And while at it, any input on oligo dt cellulose and how to use it would be very much appreciated. unsure.gif

-k81878-

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