Normalization for RNA or cDNA during two step RT-PCR? - (Apr/20/2009 )

Checking is an important step. You could also run a denaturing RNA gel to check RNA integrity, you should get clear rRNA bands

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.springerlink.com/content/p328327382188710/

Freddynb on Sep 2 2009, 01:43 PM said:

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

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

gradechips on Jun 2 2010, 11:05 PM said:

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.

hi-do you know qiashredder homogenizer is a serious need for rna extraction from macrophage or not? ihave 100000 cells and need about 100 ng rna .
thanks :hasheminia

You'd better normalize against total RNA prior reverse transcription.

Sure, cDNA clean-up enhances the PCR. In most cases, we didn't purify cDNA. Then limited volume of cDNA should be added to a PCR reaction

Hello :-)

Excuse my stupid questions.

About this RNA normalization..... Here I was given an RNA quality control protocol, you can see it.

1. What is this RNA normalization?

2. How someone could normalize the amount of mRNA if it not visible on the gel, as seen in this protocol?

3. Apart from how intact is my RNA, what more information gives me the RNA electrophoresis, if I see only the rRNA?

4. If I load my RNA sample on a 1% agarose and I don`t see anything on the level of the upper marker, would it mean that the sample is not being contaminated with gDNA?

5. As far as I undestand, non-PCR-ed gDNA makes something like.....bands with approximate size at about.....8000-10 000 bp, is that correct?

Any answer will be highly appreciated,

Nephrite