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There have been 31 items by detriar (Search limited from 23-October 19)



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#167340 Dilution series between HKG and GOI

Posted by detriar on 28 April 2014 - 12:12 AM in PCR, RT-PCR and Real-Time PCR

The efficiency is actually a property of each reaction alone. But it's hard to calculate from the single one (there are tools to do that, from each amplification curve, but they are not commonly used).

So in theory efficiency should be constant within dilution series and from several known concentration points you can calculate it.

 

Dear Trof,

actually, because I got curious about this, I re-assayed my standard, using 1:5 dilution factor. And the result came out quite good, I got slope -3.37. The first 1:5 dilution series, I think I made a pipetting mistake, or other errors.

 

Now I can use the same dilution factor between the HKG and GOI. Thank you.




#167313 Dilution series between HKG and GOI

Posted by detriar on 26 April 2014 - 02:53 AM in PCR, RT-PCR and Real-Time PCR

Dear Trof, thank you for the response smile.png but I'm sorry I don't think I get your questions, I had a terrible way to understand English sad.png, so I'm gonna explain as good as I can.

I was told to pay attention to the slope, and efficiency percentage in absolute qPCR. So when my 5-points of standard did not get the efficiency above 95%, I try to do more. I was told if 10-fold dilution factor does not work, I have to try 4 or 5-fold dilution factor for that 5 points.

For the cDNA samples, between the GOI and HKG there are no differencies in amount, so I can say that the samples are all the same. It's just differency on the dilution factor of each their standards.

Thank you smile.png




#167221 Nanodrop vs Picogreen to quantify post-PCR product

Posted by detriar on 23 April 2014 - 02:10 AM in PCR, RT-PCR and Real-Time PCR

Thank you :)




#167220 Dilution series between HKG and GOI

Posted by detriar on 23 April 2014 - 02:03 AM in PCR, RT-PCR and Real-Time PCR

Hello all,

 

I want to ask something about my qPCR assay, it's absolute quantification, and I ran the HKG first. And for the standard, I made dilution series with dilution factor 1:5. The standard curve came out good, but when I ran the standard for GOI, 1:5 dilution factor didn't come out well, so I did 1:8 dilution series, and it went good (the efficiency percentage reach 100%). So I wanna ask, is that okay to run a different dilution factor between the HKG and GOI?

 

Thank you.

 

Cheers,

 

detriar




#165699 Nanodrop vs Picogreen to quantify post-PCR product

Posted by detriar on 03 March 2014 - 07:29 PM in PCR, RT-PCR and Real-Time PCR

Dear all,

 

I wonder if it is the right subforum to ask, but since it has something to do with PCR product, I'm gonna ask here.

 

I purified my PCR product (excised the single band, and using column from roche kit to purify it) and I want to use this product as standard for the absolute quantification real-time PCR project. Thing is, I have to know the exact concentration of this purified PCR product, so that I can convert the concentration (ng/ul) to copy molecules/ul using Avogadro equation. From the journal, the researchers use picogreen to quantify that purified PCR product, but since I don't have such the equipment nor the reagen, I wonder if it is okay to quantify the purification product's concentration with Nanodrop?

 

I really hope you guys can help me with my question.

 

Thank you.

 

Cheers,

 

Dee




#165571 Different annealing temp between HKG and target gene

Posted by detriar on 26 February 2014 - 08:04 PM in PCR, RT-PCR and Real-Time PCR

Thank you Trof.




#165508 Different annealing temp between HKG and target gene

Posted by detriar on 25 February 2014 - 07:05 PM in PCR, RT-PCR and Real-Time PCR

Dear all,

 

I want to ask whether it is okay to run absolute quantification if the housekeeping gene's and target gene's annealing temperature were different. The target gene's is 65 C and the hkg gene's is 60 C.

Thank you.

 

Cheers,

dee




#164151 copies per ul to copies per gram tissue

Posted by detriar on 14 January 2014 - 07:15 PM in PCR, RT-PCR and Real-Time PCR

Thank you bob1. Appreciate it :)




#163958 copies per ul to copies per gram tissue

Posted by detriar on 08 January 2014 - 08:23 PM in PCR, RT-PCR and Real-Time PCR

Hello all,

 

I want to ask question about how to change your real-time pcr result.

 

I isolated RNA from, lets say 30 mg tissue (which the weight could be variable between one sample to another, but for example, just stick to the 30 mg). then I measured the concentration using nanodrop, and got the result 200 ng/ul (the RNA final volume is 30 ul). for the cdna synthesis, I took 100 ng total RNA, resulting in 20 ul cdna final volume, and for real-time pcr, I use 1 ul from the cdna with the final volume qpcr is 10 ul. then I ran the qpcr essay along with the standard curve, and assume all the reaction went well, I got the result 1000 copies RNA/ul.

The question is, how do I get to change the 1000  copies/ul  into x copies/mg tissue? And if mathematical equation really can make it, is it r eally okay to change that? since it's involving molecular process, not only mathematical application?

 

thank you and sorry if you find my question a lil bit confusing.

 

cheers,

detriar

 




#162657 DNA Liquid into DNA pellet

Posted by detriar on 20 November 2013 - 11:40 PM in Molecular Biology

Thank you all, it's very helpful.smile.png




#162625 DNA Liquid into DNA pellet

Posted by detriar on 20 November 2013 - 01:20 AM in Molecular Biology

Dear bob1,

is the salt considered as NaOAc? I've read it somewhere, but I can't be sure about that before I asked.

 

Thank you.




#162623 DNA Liquid into DNA pellet

Posted by detriar on 20 November 2013 - 01:11 AM in Molecular Biology

Dear all,

 

I extracted DNA from frozen tissue using Qiagen kit, and I got the final DNA result as liquid, as it was eluted with the elution buffer. But then my PI asked me to turn it into pellet. Does anyone here know how to do it, considering I've already reached the last step using the spin column and all?

 

Thank you.

Cheers,

detriar




#162620 RNA from 'bloody' frozen tissue

Posted by detriar on 19 November 2013 - 10:59 PM in Molecular Biology

Hello all,

 

I have a little difficulties with rna concentration. I extracted rna from frozen tumor tissue, grind it with liquid N2, using RNeasy Qiagen kit. Usually, I got concentration around 200-300 ng/ul, and good ratio 260/280 and 260/230 (around 2.00), but when I extracted from 'bloody' frozen tumor tissue (blood surrounding the frozen tissue), I got poor concentration (around 9-17 ng/ul). Can someone please help me with this problem? Is it my poor technique or the blood caused this?

 

Any help will do.

 

Thank you.

Cheers,

detriar




#159974 Poor efficiency standard curve

Posted by detriar on 06 September 2013 - 02:33 AM in PCR, RT-PCR and Real-Time PCR

Dear Anpu,

thanks for the response! Yes I've tried 1:5 dilution and increasing each primer to 500 nM final concentration, and the result is better. Looks like I haven't optimized the reaction well before.

 

Thank you once again.

 

Cheers, 

dee




#159877 Poor efficiency standard curve

Posted by detriar on 04 September 2013 - 12:13 AM in PCR, RT-PCR and Real-Time PCR

Hello all,

 

I have questions about standard curve for absolute quantification. I've already ran 2 times for this target gene, but I don't have any idea why the Ct between dilutions couldn't reach 3.3. From the picture I attached (please ignore Ct below 15), Ct between dilution almost reach 5 cycles! I almost sure it's not because of the pipetting (1:10 dilution, 2 ul template and 18 ul sterile ddH2O). Any chance primers caused it? Or any other aspects affecting the efficiency? I use 300 nM final conc. for each primers and 0.5 ng cDNA for 10 ul pcr final volume. I ran the housekeeping gene (the same method as this target gene), and the result is better (the slope -3.5). 

 

Any responds will be appreciated. Thank you :)

 

 

Cheers,

 

dee

Attached Thumbnails

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#159818 Pfaffl Method for relative

Posted by detriar on 02 September 2013 - 07:34 PM in PCR, RT-PCR and Real-Time PCR

I see, so it's required more tasks, that's why it's very important to set the same annealing temperature between the GOI and Ref gene from the first, eh? I'll keep that in mind if I'm about to set a new primer pair.

 

Thank you very much, bob1




#159782 Pfaffl Method for relative

Posted by detriar on 02 September 2013 - 12:09 AM in PCR, RT-PCR and Real-Time PCR

Thank you bob1 for the reply, it's very helpful smile.png. I got confused about that, but from your explanation it makes sense now.

 

Btw can you suggest me if it is okay to run the GOI gene and Ref gene not in the same experiment? What if the annealing temp between those two are different? 

 

Cheers,

 

dee




#159594 Pfaffl Method for relative

Posted by detriar on 28 August 2013 - 10:23 PM in PCR, RT-PCR and Real-Time PCR

Dear all,

 

I want to ask about Pfaffl model for relative quantification. I use the formula:

ratio = E target^(dCt target)/ E reference ^(dCt reference).

Lets say I use normal cells for the calibrator, so the dCt is Ct differences between the normal cells and the samples, right? So what is the result? Is it n-fold relative to reference gene or is it n-fold relative to normal cells?

 

And I got question about the calibrator. If I run an experiment to comparing pre- and post-treatment expression for 10 patients (each patient has both pre- and post-treatment tissue, so in total 20 samples), is it better if I use the pre-treatment tissue (tumor tissue) or normal tissue as the calibrator? 

 

Really need your help through this. Thank you :)

 

Cheers,

 

dee




#159271 100 ng RNA for cDNA synthesis

Posted by detriar on 21 August 2013 - 02:08 PM in PCR, RT-PCR and Real-Time PCR

Thank you very much, appreciate it :)




#159229 100 ng RNA for cDNA synthesis

Posted by detriar on 20 August 2013 - 09:34 PM in PCR, RT-PCR and Real-Time PCR

Hello all,

 

I hv a question, it maybe sound silly for you but I want to make sure. I extracted RNA from frozen tissues, and some give a 300-400 ng/ul concentration, but some give a very low amount RNA (15-20 ng/ul) concentrations. Since I have to do real-time PCR for these samples, I gotta normalize the RNA samples to the same concentration for cDNA synthesis. From the nanodrop result, 500 ng total RNA for cDNA synthesis is not possible for the low-amount-RNA samples (the final volume for cDNA synthesis is 20 ul), so I want to normalize all samples to 100 ng. So that's what I wanna ask, is 100 ng total RNA for cDNA synthesis considered as too low or not??

 

PS: I already have synthetized some samples using 100 ng total RNA and it went well (the qPCR can detect it) but I'm afraid I use too much component for it. E.g: the cDNA synthesis protocol said: use the final conc. 60 uM random hexamer primer (equal to 2 ul)  for 1 ug total RNA. Can I use the same amount for my 100 ng total RNA samples?

 

Oh, and I also re-run the RNA extraction for the low-amount-RNA samples but nothing change much. The concentrations are low too.

 

Thank you, any help will be appreciated!   :)

 

 

Cheers,

 

 

dee




#158202 PCR product as standard curve template

Posted by detriar on 25 July 2013 - 06:29 AM in PCR, RT-PCR and Real-Time PCR

Thanks a lot :)

Best Regards,
Dee



#158196 PCR product as standard curve template

Posted by detriar on 25 July 2013 - 05:45 AM in PCR, RT-PCR and Real-Time PCR

Dear doxorubicin and all, once again thanks for the reply. I still get curious if there is any way so I can get the final result as RNA copy number/ul?

For now I think for now I'll get those steps above done and I'll tell you how it's going :)

I have this one question though, I've read it somewhere but I have to make sure, there's no need to nanodrop the cDNA conc, right? We just need to quantify the RNA conc. of all samples and take the same amount (e.g. 100 ng) and synthesize into cDNA, then use it as templates for qPCR?



#158099 PCR product as standard curve template

Posted by detriar on 23 July 2013 - 05:55 AM in PCR, RT-PCR and Real-Time PCR

Dear doxorubicin,

Thanks for your input, I really appreciate it :)

Yes, the PI of the research wants the final result as RNA copy number/ul, because we want to check the RNA copy number of the GOI before and after treatments. But since I get a little confused abt using the PCR fragment (which is dsDNA) as standards to get the samples result in RNA copy number/ul, I think I have to ask somewhere :).

So you suggested using the one of my cDNA samples as the template for conventional PCR rather than using the cell line? Can it be done? Though my samples are tumor tissues?

Many thanks,
dee



#158080 PCR product as standard curve template

Posted by detriar on 22 July 2013 - 07:47 PM in PCR, RT-PCR and Real-Time PCR

Dear all,

I want to use my purified PCR product as template in standard curve absolute quantification. Are these steps seem correct to do:

1. I will use mcf7 cell-line cDNA as the template in conventional PCR, and the forward and reverse primers I'll use is the same with the qPCR primers. Then I'll excised the gel that contained target, and purified it using Roche Purification kit.

2. I'll quantify the concentration with nanodrop, and use the equation:
(X g/μl DNA / [pcr product length in basepairs x 660]) x 6.022 x 10^23 = Y molecules/μl

3. I'll dilute it in 10-fold serial dilutions 1:10, 1: 100,..... and use it as the standard curve template to establish copy number of the target.

The problem is, I'm not sure if these are the correct steps. My samples are frozen tissues, I extracted the RNA, and performed RT step. Then the cDNA will be used as the samples' template. If I performed the steps above, will I get the target RNA copy number or DNA copy number as a result?

On step number 1, can the same primers be used in the conventional PCR and then qPCR? As I'll use sybr green, will it affect the amplification?

And I'm not sure the equation on number 2 is correct to quantify molecules/ul. Can anyone suggests the other way to quantify it? Or if someone ever use PCR products as standard curves template?

Thanks.

Waiting for some help,

Cheers,
dee



#155600 Artificial RNA as normalizer (HK gene)

Posted by detriar on 26 May 2013 - 09:36 PM in PCR, RT-PCR and Real-Time PCR

Dear all,

I want to ask: has anyone of you ever used artificial RNA as normalizer (housekeeping gene) in real-time PCR? Because I once assayed my samples (with HK gene: GAPDH), the GAPDH Ct from pre-treatment samples and post-treatment samples are too way different. It isn't good, right? Since the HK gene should give you the same or just a bit different Ct if you run the samples with different treatment. So, someone suggested me to use the artificial RNA, but I don't know the advantages of that.

I really need some help on this problem, any advices I'll be very thankful.

Cheers,
detriar




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