Normalization for RNA or cDNA during two step RT-PCR?

Curtis 20 April 2009 - 03:13 AM
Hello all,

I have a two-step SYBR Green RT-PCR kit for RotorGene. After I extract  RNA from my samples, I proceed with cDNA synthesis right away without  measuring the RNA concentration or without normalizing equal amount of  RNA for all my samples. It is only after making cDNA that I measure the  concentration and add around 100 ng in each PCR tube.

my results are a bit funny now and I get my first signal after 35 cycles  and surprisingly the samples that should give signal sooner are always  the last ones. my friends are suggesting to measure my RNA before  proceeding with cDNA synthesis. They say maybe too much RNA in the  sample does not let cDNA to be properly synthesized !?

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public/style_images/mobile/profile/default_thumb.png littleaxt 20 April 2009 - 05:42 AM
Hi

How do you measure cDNA concentration? Do you purify it before  measureing it? Because I was told that if I measure with the Nanodrop I  would also measure the unused dNTPs and therefore I could also start  making it up :-)...


public/style_images/mobile/snapback.pngCurtis, on Apr 20 2009, 12:13 PM, said:

Hello all,

I have a two-step SYBR Green RT-PCR kit for RotorGene. After I extract  RNA from my samples, I proceed with cDNA synthesis right away without  measuring the RNA concentration or without normalizing equal amount of  RNA for all my samples. It is only after making cDNA that I measure the  concentration and add around 100 ng in each PCR tube.

my results are a bit funny now and I get my first signal after 35 cycles  and surprisingly the samples that should give signal sooner are always  the last ones. my friends are suggesting to measure my RNA before  proceeding with cDNA synthesis. They say maybe too much RNA in the  sample does not let cDNA to be properly synthesized !?

is this comment true? Reply Edit Delete   
uploads/profile/photo-thumb-6817.jpg Curtis 20 April 2009 - 06:45 AM
good point, but if I  purify the cDNa I'm gonna lose a lot of it during the purification  steps. after all we are not talking about a PCR product that has been  amplified. it's just cDNA from our RNA which is also not a lot. Reply Edit Delete   
public/style_images/mobile/profile/default_thumb.png gfischer 20 April 2009 - 07:45 AM
I always normalize the  amount of RNA I use in my RT reaction.  This way, you know that the  only source of variation in the amount of signal in the quantitative  step is from a higher level of relative expression of target RNA. Reply Edit Delete   
public/style_images/mobile/profile/default_thumb.png littleaxt 20 April 2009 - 08:48 AM
Oh, it seems possible, see http://www.biomedcen.../1471-2199/9/18 they also did it. Seems like cDNA clean-up also enhances the PCR.

But I never tried it myself, I normalize against total RNA prior reverse transcription and use relative quantification.

All the best

Jan

public/style_images/mobile/snapback.pngCurtis, on Apr 20 2009, 03:45 PM, said:

good  point, but if I purify the cDNa I'm gonna lose a lot of it during the  purification steps. after all we are not talking about a PCR product  that has been amplified. it's just cDNA from our RNA which is also not a  lot. Reply Edit Delete   
uploads/profile/photo-thumb-6817.jpg Curtis 20 April 2009 - 09:07 AM
littleaxt: yeah, relative quantification. that's what I'm doing now. the link you gave doesn't open for me.

gfischer: that would be for one-step RT-PCR? or applies to two-step as  well? how do you make sure you won't have more than 100 ng of cDNA in  your sample? because if you have more than 100 ng the amplification  might not work properly. Reply Edit Delete   
public/style_images/mobile/profile/default_thumb.png littleaxt 21 April 2009 - 12:04 AM
ok, this might help:

Libus & Štorchová 2006 “Quantification of cDNA generated by reverse  transcription of total RNA provides a simple alternative tool for  quantitative RT-PCR normalization” BioTechniques 41, 156-164

Liss 2002 “Improved quantitative real-time RT-PCR for expression  profiling of individual cells” Nucleic Acids Research 30 (17): e89

Hibbeler et al. 2008 “Housekeeping genes for quantitative expression  studies in the three-spined stickleback Gasterosteus aculeatus“ BMC  Molecular Biology 8:18

First is another idea you could try, second is a precipitation protocol  for cDNA (never tried it myself) third is another paper where they made a  cDNA clean-up prior PCR. Hope it helps, good luck!


public/style_images/mobile/snapback.pngCurtis, on Apr 20 2009, 06:07 PM, said:

littleaxt: yeah, relative quantification. that's what I'm doing now. the link you gave doesn't open for me.

gfischer: that would be for one-step RT-PCR? or applies to two-step as  well? how do you make sure you won't have more than 100 ng of cDNA in  your sample? because if you have more than 100 ng the amplification  might not work properly. Reply Edit Delete   
uploads/profile/photo-thumb-6817.jpg Curtis 21 April 2009 - 07:08 AM
ok thank you, I'll have a look now Reply Edit Delete   
public/style_images/mobile/profile/default_thumb.png Trof 22 April 2009 - 04:33 AM
We do two-step RT  real-time PCR and always measure the amount of RNA only. Usually we put  1-3 ug of RNA to the RT reaction, depending how much cDNA we need. We  don't measure nor clean-up the cDNA, use it directly to the real-time  PCR.
We can dilute the cDNA if we need more volume for more samples (like 2  times or 3 times), but usually only when using more than 1 ug. Never  exceed the 10 % of cDNA in a reaction mix. That makes from 1 ug in 20ul  RT mix, when using maximum 2ul to a 20ul PCR reaction just about 100 ng.

I think we would see it on the aplification curve if there is any  problem with high amount of cDNA, we never encountered such thing. Reply Edit Delete   
uploads/profile/photo-thumb-6817.jpg Curtis 23 April 2009 - 10:18 PM
public/style_images/mobile/snapback.pngTrof, on Apr 22 2009, 03:33 AM, said:

We  do two-step RT real-time PCR and always measure the amount of RNA only.  Usually we put 1-3 ug of RNA to the RT reaction, depending how much  cDNA we need. We don't measure nor clean-up the cDNA, use it directly to  the real-time PCR.
We can dilute the cDNA if we need more volume for more samples (like 2  times or 3 times), but usually only when using more than 1 ug. Never  exceed the 10 % of cDNA in a reaction mix. That makes from 1 ug in 20ul  RT mix, when using maximum 2ul to a 20ul PCR reaction just about 100 ng.

I think we would see it on the aplification curve if there is any  problem with high amount of cDNA, we never encountered such thing.

Thank you Trof,
your explanation was very helpful. Reply Edit Delete   
uploads/profile/photo-thumb-10294.jpg Chimp 16 June 2009 - 12:54 AM
Checking [RNA] is an  important step. You could also run a denaturing RNA gel to check RNA  integrity, you should get clear rRNA bands Reply Edit Delete   
public/style_images/mobile/profile/default_thumb.png Freddynb 02 September 2009 - 05:43 AM
I have developed a  method to amplify multiple random genes in a single real-time RT-PCR  reaction to assess quality of cDNA between samples.  It can be used to  normalize gene expression between samples.  Please see
http://www.springerl...28327382188710/ Reply Edit Delete   
public/style_images/mobile/profile/default_thumb.png sssbio 29 May 2010 - 03:32 PM
public/style_images/mobile/snapback.pngFreddynb, on Sep 2 2009, 01:43 PM, said:

I  have developed a method to amplify multiple random genes in a single  real-time RT-PCR reaction to assess quality of cDNA between samples.  It  can be used to normalize gene expression between samples.  Please see
http://www.springerl...28327382188710/

Hi,

I need your help regarding quantification of mRNA levels of trageted  genes in control and treated samples. I have no choice of reference  housekeeping genes to be used for relative quantification as these genes  are showing significant down regulation in treated samples. So what are  other methods that I can use to quantify mRNA levels?

I will be very happy if you guide me for this.

Thanks Reply Edit Delete   
public/style_images/mobile/profile/default_thumb.png gradechips 02 June 2010 - 11:05 PM
Please help me, I have  been doing RT-PCR for the past six months and i still have problems.  During this period i was only able to get decent Cts for the house  keeping gene (18) twice but all the other times the Cts for both  GAPDH/beta actin have been really high 25 or inconsistent within  triplicate samples (20, 24, 29). Obviously i cant use these data to  analyze gene expression and am getting frustrated. After extracting RNA  using Trizol i quantify it by ODing and i usually get ~2.500ng/ul and i  use 2ug for the cDNA synthesis and i normally dilute the cDNA 1:50. My  lab mate normally gets low and consistent Ct for the house keeping gene  using the same reagents. I have been trouble shooting for the past 6  months with his help but am not getting it.
Is it because vortexing shear my samples? Please someone help me.
Thanks in advance Reply Edit Delete   
public/style_images/mobile/profile/default_thumb.png Nataliya 24 June 2010 - 01:19 AM
public/style_images/mobile/snapback.pnggradechips, on Jun 2 2010, 11:05 PM, said:

Please  help me, I have been doing RT-PCR for the past six months and i still  have problems. During this period i was only able to get decent Cts for  the house keeping gene (18) twice but all the other times the Cts for  both GAPDH/beta actin have been really high 25 or inconsistent within  triplicate samples (20, 24, 29). Obviously i cant use these data to  analyze gene expression and am getting frustrated. After extracting RNA  using Trizol i quantify it by ODing and i usually get ~2.500ng/ul and i  use 2ug for the cDNA synthesis and i normally dilute the cDNA 1:50. My  lab mate normally gets low and consistent Ct for the house keeping gene  using the same reagents. I have been trouble shooting for the past 6  months with his help but am not getting it.
Is it because vortexing shear my samples? Please someone help me.
Thanks in advance


I usually use RNeasy Mini Kit (50)  and RNase-Free DNase Set (50) from  Qiagen. Once I have got RNA extracted I use it for cDNA synthesis or  keep at -70 C. For cDNA synthesis I used SuperScriptTM III First-Strand  Synthesis System for RT-PCR (Invitrogen, Cat. No: 18080-051). For RT-PCR  I diluted cDNA 1:5. Sometimes I measured cDNA by nanodrop and the  concentration of the diluted cDNA samples was around 200 ng/ul I have  never faced the problem with low values for house-keeping genes. I  normally get 13-14 Ct for GAPDH. Probably, you don't need to dilute  samples up to 50 ul/ml.