Can anyone please tell me what is the level of expression of a single copy gene (36B4) compared to telomere in a cell? I mean, all cells might have different level of expression of both genes, but when we are using the same sample, it can be relative right?
My problem is that I finished amplifying my telomere in the sample and they amplified very well at Ct of between 12 to 21. But, in case of single copy gene it is not showing amplification even at the Ct of 18. I use the same thermal profile and same constitutents in the master mix as well. The sample that I used has the same concentration of 100 picogram per ml to amplify both telomere and single copy gene and I expect that single copy gene should express at earlier Ct than telomere at least in both diseased and non-diseased samples. Is that right what I am thinking or have understanding?
If single copy gene is not amplifying at earlier Ct than telomere then do you think there is something wrong with anything in my process of constituents of masters mix? If so, what it might be?
I understand that single copy gene should amplify at lower Ct than telomere because single copy gene is a reference gene. What do you think?
Hi Rajesh
In my opinion,..
1) you should first try the amplification of your gene of interest in a regular PCR reaction to see if everything is fine or not...
2) if this gene has been used by anyother else earlier see the literature to get information about the copy number of this gene...
3) if no information is availabe..use different concentration of template first to see what would be appropriate to study the expression of this gene..
4) I am still confused about 'amplification of telomere' and 'expression' of these two genes......Is telomere 'expressed'.......could you explain what exactly you are trying to do in this experiment?....
5) Are you doing real-time PCR using DNA or RNA as template....
6) I suggest....as in the begining.....optimize everything in regular PCR.......and ...........if everything works in the regular PCR...then only you should go ahead and do the 'Real-time PCR'....
bye
Ajay, I am sorry for being so late. I have been busy fixiing my reaction. Finally, I have been able to fix my reaction. Now, comign back to the question.
In fact, telomere is the repetative sequence of (TTAGG)n at the end of the DNA. This is non-coding sequence. its function is just to protect DNA from being degraded. It is not a gene. It doesn't encode any protein or something. Howoever, its lenght is being mantained by another mass of complex proteins which contains 6 different types of proteins which make up one single copmlex called SHELTERIN COMPLEX. There are genes which encodes for those proteins. It is also called TELOMERASE PROTEIN. There is genes for telomerase protein.
Hi rajesh , im trying the qrt-pcr by using 36b4 single copy gene .As ajay said i tried first with normal pcr and gort annealing temp.at 60 degrees.so ,further i proceeded with qrt-pcr.But,my problem is im not getting NTC for this.if NTC comes only i will be able to proceed for quantification.can u tell me the reason
Hello Udaya,
I think I can. But, can you please tell me what do you mean by "not getting NTC". I think NTC is for "NO TEMPLATE CONTROL". That means you should add water instead of any DNA template in that reaction. It is for negative control and we have to run it to observe that any contamination is there or not -- just to be sure. I guess, you might already know about this. So, can you please elaborate your problem?
In fact, we should not see any amplification in NTC reaction.
hi rajesh.s ur right we cant run the NTC with c.DNA.As u said i ran the NTC WITH FRWPRIMER+ REVERSE PRIMER+D.WATER+SYBR .As i said im not getting NTC means .we have 2 get NTC result with undetermine value know.so that value im not getting
and moreover im keeping at 95 -10 min
95-15 sec
40 cycles
60 -1 min
followed by dissociation step.
rajesh,we need to conform the NTC result before we r going with c-DNA amplication when we r using qRT PCR,now i think u understood my pblm
tqqq
Okay Udaya,
Let me guess here. The first thing is that you are mentioning about cDNA (comlimentary DNA) which means you are not directly measuring the length of telomere becuase telomere is a non-coding sequence. I guess, you are trying to measure the expression-level of telomerase gene, right? Second thing is that, you are right when you say that you have to confirm with the result of NTC, but it has to be in the same plate in which you run the cDNA so that you can be sure that your technique is right when it comes to getting contamination in your samples.
Now, I am going to point out few things according to what I have understood from your question:
1. You have to run the cDNA of telomerase gene in the same plate parallel to the well containing reaction mixture for NTC and positive control.
2. Second, when we are doing real-time PCR (which ever real-time PCR) -- either reverse transcriptase real-time PCR or absolute quantification, we never expect to get any kind of expression or amplification from the well containing NTC reaction mixture.
Now, follow the following things and take care of these steps:
1. Check for the concentration of your cDNA using nanodrop machine and make sure you have enough sample to run PCR.
2. Make sure you are not using the final reaction volume in each reaction below what is recommended. Usually, we use 20 or 25 micro liter for the optimization reaction. I have seen people using as less as 10 microliter which is not a good practice and it is quite obvious that you might not get result or even good quality result with that much final volume of the reaction -- because the lower your final volume is, the greater is the chance of pipetting error which is inevitable.
3. Usually we do not consider any amplification over 30 reaction cycle otherwise stated.
4. Make sure to get as much lower Ct value as you can -- preferiably something between 7 to 13 Ct cycles.
5. Run your reaction in duplicate or preferriably triplicate to make sure of your techniques and compare the Ct values of those replicates (duplicate or triplicate).
6. While preparing reaction mixture, make sure you mix it well and go through brief spining before you transfer your sample in the reaction -- I mean your cDNA sample to the reaction mixture.
7. Use SYBR green with ROX dye (a reference dye) -- this will minimize any chances of background noise in the reaction.
8. Since the well containing NTC reaction do not have any kind of template, it will never show any amplification unless there is contamination or formation of primer dimer.
9. Use the primer being used by Cawthon, but make sure how he used it. I will soon send you his paper.
10. Since you are performing your reaction with cDNA, therefore, I guess that it is nothing about absolute quantification. So, in your case you have to calculate the expression level using delta-delta Ct method. Look for it or let me know if you can't find it. I will send you the link.
At last, I am again retirariting that you should not get any value in NTC. It is fine that you are not getting any amplification value. If you are not getting any value, you are good to go with your cDNA. Just run another plate containing replicates of cDNA reaction along with postiive and negative control (which is NTC).
Let me know if you are further experiencing any problem with the reaction and wait for my next message with some papers.
yes you are right Rajesh.Every point im following .but im trying with 30cycles.Anyway thank you for your valuables suggestions.They will be consider able.Thank you onceagain.please send me the links regarding this single copy genes.See you.
Another one is here. This one is the first paper by Cawthon, I guess.
Hi Rajesh,
I am working on 36B4 gene as reference gene in one of my experiment. But I am unable to get an amplified product neither at 60 degrees or at 72 or both ( as suggested in literature).
Can you please share how did you resolve your problem...I am using same pair of primers as suggested by Cawthon, 2004.
- Badges
- Science method