Molecular Assays

We have the capabilities to provide molecular biology based services for the isolation of Eukaryotic and Prokayrotic (Bacteria, Yeast, Plant, Baculovirus, Tissue Culture Cells, Primary Tissues):


  1. Total RNA
  2. mRNA
  3. cDNA Purification for Microarrays

Real-Time qRT-PCR

(Real-Time Quantitative Reverse Transcription PCR) is a major development of PCR technology that enables reliable detection and measurement of products generated during each cycle of PCR process. This technique became possible after introduction of an oligonucleotide probe which was designed to hybridize within the target sequence. Cleavage of the probe during PCR because of the 5' nuclease activity of Taq polymerase can be used to detect amplification of the target-specific product.


How It Works:

Courtesy: http://www.ncbi.nlm.nih.gov/probe/docs/techqpcr/

Techniques to monitor degradation of the probe

  1. Intercalation of double-stranded DNA-binding dyes
  2. 32Pprobe labeling
  3. Labeling of the probe with fluorescent dyes

TaqMan
assay (named after TaqDNA polymerase) was one of the earliest methods introduced for real time PCR reaction monitoring and has been widely adopted for both the quantification of mRNAs and for detecting variation. The method exploits the 5' endonuclease activity of Taq DNA polymerase to cleave an oligonucleotide probe during PCR, thereby generating a detectable signal. The probes are fluorescently labeled at their 5' end and are non-extendable at their 3' end by chemical modification. Specificity is conferred at three levels: via two PCR primers and the probe. Applied Biosystems probes also include a minor groove binder for added specificity.

Applications of Real Time Quantitative RT-PCR

  1. Relative and absolute quantification of gene expression.
  2. Validation of DNA microarray results.
  3. Variation analysis including SNP discovery and validation.
  4. Counting bacterial, viral, or fungal loads, etc.


Courtesy: http://www.ncbi.nlm.nih.gov/probe/docs/techqpcr/

Nomenclature used in RT-qRT-PCR

Baseline
is defined as PCR cycles in which a reporter fluorescent signal is accumulating but is beneath the limits of detection of the instrument.

ΔRn
is an increment of fluorescent signal at each time point. The ΔRn values are plotted versus the cycle number.

Threshold is an arbitrary level of fluorescence chosen on the basis of the baseline variability. A signal that is detected above the threshold is considered a real signal that can be used to define the threshold cycle (Ct) for a sample. Threshold can be adjusted for each experiment so that it is in the region of exponential amplification across all plots.

Ct is defined as the fractional PCR cycle number at which the reporter fluorescence is greater than the threshold. The Ct is a basic principle of real time PCR and is an essential component in producing accurate and reproducible data.


RNA Extraction, cDNA synthesis and Quantitative PCR:

Total RNA can be isolated from cells in culture or from tissues. RNA can be extracted using Tri-reagent methodology or preparatory RNA isolation kits which typically use a combination of organic and solid phase extraction methodologies.


Either Tri-reagent or a lysis buffer are used for the extraction of RNA samples, which are finally resuspended in Tris-EDTA buffer. The RNA mass is determined using an NanoDrop, purity assessed from the 260/280nm and 230/260 ratio. Following extraction, RNA is processed with a Turbo DNA-free kit to reduce any residual genomic DNA (gDNA) contamination prior to reverse transcription and production of copy DNA (cDNA) using the High Capacity cDNA Reverse Transcription Kit.


Once formed cDNA samples can be probed using quantitative PCR (QPCR) and commercially available TaqMan probes from Applied Biosystems, currently there are over 8 million predesigned TaqManTM assays available .


Typically QPCR require a suitable endogenous control - a stable gene that can be used to make relative measures against. Commercially available endogenous control TaqMan probes are available such as beta-glucuronidase (GUSB), hypoxanthine-guanine hosphoribosyltransferase HPRT1 (HGPRT), beta-2-microglobulin (B2M) and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Selection of an appropriate control requires screening cDNA for a range of endogenous controls and analysing the data using GeneNorm. Once identified endogenous control genes can be determined alongside a gene of interest to determine changes in gene expression. This enables normalising of target gene expression levels using the equation 2(-ΔΔCt), where ΔΔCt = ΔCt (treated) -ΔCt (control; media) and this calculates fold-change in the gene expression.


A dual assay methodology is used which which enables endogenous control genes and genes of interest to be investigated simultaneously on a sample in a single well - this reduces the quantity of sample required. QPCR experiments are performed on a StepOne Plus instrument using a thermal profile of 2min at 50οC, 10min at 95οC, 50 cycles of 15s at 95οC and 1min at 60οC. Non-template controls were included for all samples.


Non-template controls (NTCs) are determined for all samples and represent a negative control which is run in parallel for each sample, in which the reverse transcriptase enzyme, which converts mRNA to cDNA, is removed. If there is amplification in NTCs it represents genomic DNA contamination and not mRNA expression.