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Questions related to Quantitative RT-PCR
Hi everyone, I am carryied out a GWAS with the sample size about 5000 control versus 5000 cases, and 260,000 variants at the beginning After the quality control with MAF at 0.05, I achieved only...
12 December 2019 6,751 3 View
What can I do when I always have BADROX in my qPCR measurements? The multicomponent plot of the ROX seems like in the picture. Does it occur some problem in the evaluation if I ignore ROX and the...
29 November 2019 6,944 0 View
Hello! I ran my first qPCR but I forgot to dilute the cDNA before running. I started with rtPCR of 2000ng of RNA in 20ul reaction volume. My RNA purity was great and I included a RNAse inhibitor....
19 November 2019 3,166 3 View
I submitted my plant RNA samples to be run on an Agilent Bioanalyzer 2100 and received back the results. Most of the extraction kits I tested were good in quantity (ranging from 40-270ng/uL) as...
23 October 2019 5,513 5 View
Hello, I'm conducting siRNA-mediated gene knockdown experiment, and after 48 hours, I re-transfected again because I will be collecting cells 72 hours after seeding cells. However, I noticed...
07 September 2019 2,990 4 View
Input: - RT QPCR dataset row CT values interpolate corrected - 50 target genes - 10 reference genes - 3 different treatments with 3 replicates each - 1 control with 3 replicates Objective: -...
20 August 2019 1,678 3 View
I am trying to run a TaqMan assay where my housekeeping probe is labelled with VIC. I do not have access to a qPCR machine with VIC specifically defined, but HEX is recommended as a substitute. Is...
19 August 2019 9,317 4 View
Im new to DEGs and PCR data and i received a RT-qPCR data of interpolate corrected CT values of around 50 microRNAs . The dataset contains 5 different conditions each condition contains : - 3...
17 August 2019 3,520 3 View
Hi everyone, I am conducting an experiment to detect copy number variation among my gDNA samples using RT-PCR. I have generated standard curve for the genes of my interests and two controls...
24 July 2019 6,984 3 View
I did chromatin extraction using both commercial kit and homemade recipe. By using RNase A and protease K digestion together with phenol-chloroform-isoamyl or on-column cleaning, I then isolated...
23 July 2019 687 2 View
I am currently validating 18S rRNA, HPRT1 and GAPDH as housekeeping genes to normalize data obtained from my case:control qPCR study using human peripheral blood. Group 1 - Patients (cases)...
22 July 2019 586 3 View
I need to perform qPCR detection of specific fungal species in plant (strawberry). I need to include internal control (housekeeping gene) for plant, I have found that some people use COX gene, but...
12 July 2019 7,841 2 View
I aim to compare the transcription level of two genes that encode two enzymes with the same function. I extracted mRNA and compared their transcription using RT-PCR. The Ct (also called Cq) values...
06 July 2019 8,310 9 View
If you have you used the QX100 or QX200 system, or perhaps another digital PCR system, what do you think? What would you improve? What are some frequently asked questions?
01 July 2019 5,994 5 View
I am attempting to perform a qPCR standard curve using viral DNA (Adenovirus and MCMV) and cDNA (Influenza A) to be used to determine relative abundance of these viruses in primary samples. Viral...
25 June 2019 481 4 View
I'm developing a MethyLight assay to verify a specific CpG site in a promoter region of my interest gene in rats. Unfortunately, I don't have a lot of choices to design the probe and primers,...
21 June 2019 1,085 3 View
The RIP experiments include input, IP of IgG, and IP of target antibody, how to deduct background signal for RIP-seq analysis? Do the signals in the IgG group or the input group should be deduct...
12 June 2019 8,002 0 View
Good Morning, I have a problem with qPCR measurements and I hope you can help me with that. We would like to implement a probe (TaqMan) assay. For receiving our samples, we isolated neutrophils...
07 June 2019 6,125 40 View
Hi Everyone, I have been working with a FAC sorted population of embryonic brain cells. I typically get between 50-90% viability following the sort but only 12 000 - 20 000 cells. I have been...
24 May 2019 4,272 5 View
I have a RotorGene Q (Qiagen) and I'll perform qPCR reaction for relative quantification. I have SYBR green I 10.000x and the other components of the reaction. Can I make a homemade master mix?...
21 May 2019 10,022 3 View
In my HRM analyzes, although I use the same samples in the same analysis, I get different Tm values. And I've repeated it many times, but there has always been a discrepancy. For example, even...
16 May 2019 4,848 10 View
Hello! I'm trying to perform a TaqMan qPCR to check for certain fish species in environmental DNA samples. I am using a published primer + probe set. The probe is carrying FAM at it's 5' end and...
15 April 2019 4,998 15 View
Hi, I am using geNorm, Normfinder, RefFinder, and BestKeeper to identify and select the appropriate reference genes for the normalization in the RT-qPCR analysis. I was trying to identify the...
09 April 2019 6,758 3 View
Hello I did automated DNA extraction from cervical swabs samples and for this is did elution in 100 microliter but at the end of DNA extraction I got very low DNA concentration which is like...
07 April 2019 8,167 5 View