Understanding real-time PCR terminology--Taq Talk Episode 1

[Music] welcome to the applied biosystems tech talk video series where we break down real-time pcr to make it easier for you real-time reverse transcription polymerase chain reaction phew that was a mouthful how about rtq pcr well that's certainly easier to say but how exactly did we get from there to rtq pcr well that's a great question let's talk through it and discuss some of the common terminology and acronyms that you will encounter when using this technology let's start with an easy one polymerase chain reaction that's our good friend pcr okay what about rt like many people you may assume that rt is short for real time rt is actually an acronym for reverse transcription rt pcr or reverse transcription pcr is a type of pcr in which rna serves as the starting template the rna is transcribed into complementary dna or cdna by a reverse transcriptase enzyme the cdna is then used as a template for traditional pcr since we're already talking about reverse transcription let's clarify what we mean by one step or two-step rt-pcr in one-step rt-pcr the reverse transcription and pcr reactions are carried out in the same tube in two-step rt-pcr reverse transcription is done first as a separate reaction after that a portion of the c-dna is added to a pcr mix for amplification that leaves us with q how did we get from real time to q in real time pcr the instrument detects a fluorescent signal that is generated during target amplification in each pcr cycle in real time the data is then analyzed to determine the relative or absolute quantity of the target sequence in the original sample in other words the data is collected in real time to obtain quantitative results and that's how quantitative or q became synonymous with real time we should also talk about rn and cq or ct values but we need to define baselines and thresholds before we do that a qpcr baseline is the low level fluorescence emitted during early pcr cycles it represents the background signal or noise the target threshold which appears as a horizontal line in the amplification plot is the signal intensity that reflects a statistically significant increase over the baseline the threshold is then used to determine the cq or quantification cycle the cq value corresponds to the pcr cycle in which the sample amplification curve crosses the target threshold this is where the fluorescent signal exceeds background fluorescence which indicates true amplification a low cq correlates with a high target expression level in a sample over the years cq has also been referred to as a threshold cycle or ct crossing point or cp and takeoff point or tlp they all mean the same thing but quantification cycle is preferred according to mikey guidelines to ensure consistency you may run across a few other terms during data analysis anytime this little triangle or delta appears next to a value it indicates the value has been calculated as the difference between two other values for example delta ct or dct is the difference between the ct values of your target gene and your endogenous control delta delta ct or ddct is calculated during a comparative ct analysis in which the normalized delta cts of a test sample and a reference sample are compared similarly delta rn is the rn value of an experimental reaction minus the rn value of the baseline signal maybe you're wondering what rn means rn stands for normalized reporter it is the fluorescent signal from the reporter die normalized to or divided by the signal from a passive reference die now that you're an expert in real-time pcr terminology check out some of our other applied biosystems tech talk videos and our online learning center for more in-depth information about this technology and its applications [Music] you

Channel: Thermo Fisher Scientific

Views: 21,184

Keywords: accelerating drug discovery, biomarker discovery, biomarker validation, data quality, gene expression, genetic analysis, genotyping, graduate student, imnoncology, lab manager, learning qpcr, miqe guidelines, neuroscience, oncology, optimizing drug discovery, pcr, post-doc, principle investigator, publishing tips, qpcr, qpcr acronyms, qpcr assays, qpcr instruments, qpcr master mix, qpcr primers and probes, qpcr terminology, qpcr terms, real-time pcr

Id: pL2gW6VX_Js

Length: 5min 16sec (316 seconds)

Published: Thu Oct 14 2021