Ct value of housekeeping gene - (May/03/2014 )
I am using Bio-Rad iQ5 for relative quantification of mRNA of a gene in Klebsiella pneumoniae. 16S rRNA is used for normalization. When I use 10 x diluted cDNAs, Ct values of 16S rRNA ranges between 6 and 8 while those of gene of interest fall in the range of 24 -25 for control and 16 -17 for experimental samples.
Should I use cDNA 10x diluted for gene of interest and 100x or more diluted for 16S rRNA. If yes then how can I do calculations to find out mRNA level.
Dr Soumble Zulfiqar
Ribosomal rRNAs are known for being problematic reference genes, due to their very high relative abundance.
Commonly the primer concentrations for ribosomal rRNA is limited (i.e. lowered) in comparison to gene of nterest primers, so it doesn't give such small Cts.
Ct below 15 in not usable for quantification. You may use more diluted cDNA (50x seems optimal from your gene of interest Cts), but that won't help your 16S Cts much unless you limit the primers, 10-fold dilution only moves Ct around 3, so you would still have Cts around 9-11, which is too low.
Generaly housekeeping gene and gene of interest should have similar abundancies. As mentioned, rRNAs have rarely abundance comparable to normal genes even with limited primers. It's also possible to look for oher housekeeping genes, though that may be difficult in bacteria.
Dear Trof
Thank you so much for your reply. Mean while, I have also tried GyrA and Mdh as house keeping genes. Ct values of GyrA and Mdh are compatible for those of gene of interest. But when I did real time of 16S rRNA, GyrA and Mdh for various samples simultaneously, and calculated the expression level of GyrA and Mdh with respect to 16S rRNA considering it (16S rRNA) as housekeeping gene only, the results were as follows:
Sample ID
Mdh
GyrA
A
1
1
B
1.61726
0.36667
C
3.4904
0.06904
D
4.70742
0.09185
E
1.42505
0.31052
F
1.58548
0.29523
G
0.3846
0.08292
Sample ID 'A' was used as control. All others (B-G) were experimental
Ideally all these values should be 1 as all genes are considered as house keeping genes i.e., there level remains same in cells irrespective of any environmental factor.
Now I don't know which gene should I use as reference in Real Time PCR. Will you kindly suggest me what to do.
Dr Soumble
Actually no gene is stable at any conditions. Some genes are used as if they never change, but they do. People just don't bother to check if that's true for their situation and they never find that their results ar invalid.
The only way is to test several "housekeeping" genes and find those most stable at your specific conditions.
More resources about this complex topic in this thread:
http://www.protocol-online.org/forums/topic/32220-reference-genes-selection-and-validaton-for-qpcr/
I have gone through reading material, you recommended. I really got very valuable information. Now I have three points to discuss:
1. I used Norm finder to find out most suitable reference gene out of 16S rRNA, Mdh and GyrA (These have been used as reference gene and reported in different papers). One thing is not clear i.e., what is meant by input data. This can't be Ct values as in example figures are in 1000s that isn't in range of Ct values. Similarly This can't be fold change in expression level because to calculate it first we need some reference gene. Please keep in mind that I don't have standards for absolute quantification, rather I am doing comparative analysis. Can you help me in this regard?
2. I use 2 μg RNA (quantified before DNase treatment) for cDNA synthesis. Now I have planned for:
RNA quantification after treatment and then use of same amount of RNA for cDNA synthesis.
cDNA quantification and then use of same amount of cDNA for real time PCR for three candidate reference genes (16S rRNA, Mdh and GyrA) .
Among these that gene will be used for normalization whose Ct values will be similar among samples obtained from different environmental conditions.
3. If the primer concentrations for ribosomal rRNA is limited, should it be taken into consideration during calculations (data analysis).
I'll be grateful having suggestions about above mentioned points.