How To Do Real Time Gene Expression Work for Beginners?

This gene  expression experiment was done using  rice Oryza sativa seeds at various stages of seed  development. The following picture will show you the flow of the work.

Shoot and seed samples of the rice plant were collected and  immediately  frozen in liquid nitrogen. Futhermore, RNA extraction was done using Trizol method with slight modifications like twice the addition of Lithium Chloride precipitation. After that RNA quality and quantity was verified using gel electrophoresis  and nanodrop spectrophotometer. Two intact bands(18s and 28s) of RNA were observed  indicating a good quality RNA. Next the crude RNA samples were treated with Promega RQ1 RNase free DNase.

 For the purpose of clarity, I posted here the picture of the different stages of grain development in rice.

Here are the different tissues we considered for the experiment.

DOWNSTREAM APPLICATIONS
    cDNA synthesis

  Afterwards, cDNA was synthesized   from  the DNase treated RNA using the Roche 1^st Transcript cDNA synthesis kit. Here are the components of the reaction: template mixture( oligo dT, random hexamer, DNase treated  RNA) and reverse transcriptase mixture( RT buffer, RNase inhibitor, Deoxynucleotide mix and reverse transcriptase).
Here is the procedure how to do it? First, denature the template mixture for 10min at 65 degrees centigrade and put  immediately in ice for 5mins. Then followed by addition of  the RT mixture to the template mixture. Incubate for 10 min  at  25 degrees centigrade then   followed by 30 min at 65 degrees centigrade.

PRIMER TEST

Primer design and primer check took most of  my time in doing  this work. For the purpose of protecting my Intellectual Property rights, I would not reveal the identity of the genes which I designed and tested. Our research will be published officially this year.

So here is the point in this work.
First, you have to design your primers using bioinformatic softwares. I would discuss in detail how to design primers in my next post. After designing my primers and ordering them, we then test our primers with cDNA using the conventional PCR like G-storm cycler to check PCR product size and the number of band(s) that appeared on the gel. If the primers showed to have one distinct band  and  appropriate product size on the gel, I can now proceed in performing the real time PCR primer check (qRT-PCR). qRT-PCR is highly sensitive and very accurate. In the illustration, after performing melting peak analysis the primers which showed to have two peaks were discarded and  we JUST considered  the primers with ONLY ONE MELTING PEAK . So in short, primers working with conventional PCR will not necessarily work with qRT-PCR. Primer design and specificity is the first priority. 

SELECTION OF REFERENCE GENES

From an original choice of  four reference genes(housekeeping genes), we selected three reference genes to normalize the expression of our target genes based on its expression profile using its Crossing point. The more uniform the crossing point(Ct or Cp) to each other the better it is. Gene stability was checked across the different rice genotypes and its accompanied rice tissues.

We tested the reference genes stability at the different stages of grain development and across different genotypes of rice. We used the geNorm software to evaluate our four reference genes which debunks or removes reference genes with M(relative stability)>1.5 which in this case it was EP1(Ref4). Bioinformatic softwares like geNorm will make the work very easy. Just click on the link.

NOTE:

Contamination should be avoided with all means possible. Always work in  a room designated for RNA extraction only and a room with no possible contamination of plasmids.

 Relative expression computation

Computation of relative expression could be done by means of relative quantification with of course the use of internal reference genes(control genes) and the other method utilizes the power of calculus with is the absolute of quantification method. Pfaffl 2001 suggests the used of an internal calibrator/control sample to normalize the expression of the target sample using the formula of delta delta Ct(relative expression).Relative expression computation can be done in Microsoft excel.  Another method used to compute was through delta Ct method wherein all Ct values of samples or reference genes were converted to raw expression values with used of a control sample or a sample with highest(earliest Ct). These raw expression of the target genes would be normalise by the geomean of AT LEAST three reference genes in order to correct its raw expression (GeNoRM, Vandonsempele, 2000).

Here is the sample gene expression  data we got using relative quantification method  with the use of  at least THREE reference  genes.

Reference:
Pfaffl M.W. , 2001. A new mathematical model for relative quantification in real-time RT–PCR 

Vandosempele et al 2002