Quantitative Real-Time PCR
Quantitative PCR (qPCR) is used for DNA quantitation such as viral load. Most applications use qPCR for the quantitation of RNA levels – more specifically, reverse-transcription quantitative PCR (RT-qPCR) – to measure the levels of mRNAs, miRNAs and other RNA species.
The NASR operates four Applied Biosystems 7900HT Fast Real-Time PCR Systems. Key applications include SNP detection using the fluorogenic 5’ nuclease assay (see genotyping) and gene expression quantification (absolute and relative). TaqMan™ and SYBR Green chemistries are supported.
Quantitative Real-Time PCR includes:
- TaqMan Gene Expression Assays
- TaqMan Low Density Array (TLDA)
- TaqMan MicroRNA Assays
- Digital Multiplexed Gene Expression Analysis Using the NanoString nCounter System
- Validation of Microarray Results
- SYBR Green Chemistry
- Data Analysis
TaqMan Gene Expression Assays
This method capitalizes on a quantitative relationship between the amount of starting target sample and the amount of PCR product at any given PCR cycle number.
The 5’ nuclease chemistry exploits the exonuclease activity of AmpliTaq Gold DNA polymerase by using a cleavable fluorescent probe in combination with forward and reverse PCR primers.
TaqMan Assays have the highest specificity, highest sensitivity and the largest dynamic range of any gene expression technology.
TaqMan Low Density Array (TLDA)
For more throughput, TaqMan Gene Expression Assays are available pre-loaded onto the TaqMan Low Density Array, a 384-well microfluidic card that enables one to eight samples to be run in parallel across 12 to 384 targets, without the need for liquid handling robotics.
Additionally, the NASR uses arrays customized with genes, pathways or prognostic panels with as few as 12 genes or as many as 380. Combined with a pre-amplification step, very small amounts of samples, as well as archived tissue samples such as laser-capture microdissection (LCM), formalin-fixed paraffin-embedded tissues (FFPE) and needle biopsies, can be processed for gene expression analysis.
TaqMan MicroRNA Assays and Arrays
TaqMan MicroRNA Assays incorporate a target-specific, stem-loop, reverse-transcription primer. The new primer design addresses a fundamental problem in miRNA quantitation caused by the short length of mature miRNAs (~22nt), which prohibits conventional design of a random-primed RT step followed by a specific real-time assay. The stem-loop structure provides specificity for only the mature miRNA target and forms a RT primer/mature miRNA-chimera that extends the 5’ end of the miRNA.
The resulting longer RT amplicon presents a template amenable to standard real-time PCR using TaqMan Assays. These assays are not only specific for mature miRNAs, but also can distinguish among highly homologous targets.
TaqMan assays are known for a wide linear dynamic range, up to nine logs, and a miRNA profile can be generated in as little as three hours compared to several days for microarrays.
TaqMan® MicroRNA Arrays provide all the advantages of TaqMan® MicroRNA Assays in a convenient, pre-configured microfluidic card minimizing experimental variability and effort required to run 384 TaqMan MicroRNA Assays in parallel.
TaqMan® MicroRNA Arrays v3.0 is a pre-configured 2 micro fluidic card set, A and B, with currently 754 unique assays specific to human microRNAs updated to capture new content available in Sanger miRBase v14. In addition, each array contains four control assays, three selected candidate endogenous control assays and one negative control assay. An optional preamplification step can be included for small amounts of sample.
Digital Multiplexed Gene Expression Analysis Using the NanoString nCounter System
NanoString’s novel digital technology uses color-coded, fluorescently labeled molecular barcodes and single molecule imaging to detect and count hundreds of unique transcripts in a single reaction. Bypassing the need for amplification and reverse transcription eliminates enzymatic bias and simplifies the nCounter workflow to consist of hybridization, post-hybridization processing and digital data acquisition. The complexity of the barcodes, containing one of four colors in each of six positions, allows a large diversity of targets present in the same sample to be individually distinguished during data collection.
The NASR has operated the nCounter System from NanoString Technologies since January 2010. In addition, by the end of March of 2010, the OSUCCC NASR completed beta testing for a new product for microRNA analysis that uses multiplex digital barcode technology to profile the human miRNA transcriptome in a single tube.
The current applications include:
Gene Expression Analysis: NanoString’s gene expression CodeSets are pairs of approximately 50-bases called capture and reporter probes that hybridize to the RNA sample and are used to detect and count mRNA transcripts. The reporter probe carries the signal and the capture probe is used to immobilize the complex for data collection. After hybridization, samples are transferred to the nCounter Prep Station where excess probes are removed and probe/target complexes are aligned and immobilized in the nCounter Cartridge. Cartridges are then placed in the nCounter Digital Analyzer for data collection. This technology can detect and count hundreds of gene transcripts simultaneously with a sensitivity of less than one copy per cell. The system can directly assay tissue and blood lysates as well as FFPE extracts with protocols starting from 100ng or less of total RNA. mRNA expression analysis can be performed simultaneously on up to 800 genes for validation and routine testing of whole-genome screens, for the quantification of entire pathways, biological processes, or prognostic signatures in a single tube.
miRNA Expression Analysis: The NanoString miRNA assay uses an additional sample processing step to enable the detection of small RNAs. Preparation of small RNA samples involves the ligation of a specific DNA tag onto the 3’ end of each mature miRNA; these tags are designed to normalize the Tm’s of the miRNAs as well as to provide a unique identification for each miRNA species in the sample. The tagging is accomplished in a multiplexed ligation reaction using reverse-complementary bridge oligonucleotides to direct the ligation of each miRNA to its designated tag. Following the ligation reaction, excess tags and bridges are removed, and the resulting material is hybridized with a panel of miRNA:tag-specific nCounter capture and barcoded reporter probes. Following purification, each captured barcode is counted and tabulated in the nCounter assay. NanoString miRNA assay can be used for discovery experiments because the entire micronome can be covered. Both nCounter human and mouse miRNA expression assay kits are available. 100ng or less of total RNA per sample is used for one profiling reaction.
Copy Number Variation Analysis: The nCounter Copy Number Variation CodeSets provide everything needed to interrogate up to 800 regions of the human genome in a single multiplexed reaction with as little as 300ng purified genomic DNA of starting material. The nCounter Custom CNV Assay is based on the standard nCounter assay with two important additions: DNA fragmentation and denaturation. These two steps yield single-stranded targets for hybridization with nCounter probe pairs, the reporter and capture probes, following the nCounter workflow including hybridization, post-hybridization processing and digital data acquisition. Each CNV probe pair is identified by the “color code” generated by six ordered fluorescent spots present on the Reporter Probe. The Reporter Probes on the surface of the cartridge are then counted and tabulated.
nCounter Human Karyotype Panel: The Human Karyotype Panel consists of 338 probes spanning all 24 chromosomes at a rate of approximately 8 probes per chromosome arm. This coverage enables highly accurate confirmation of diploidy and identification of aneuploidies for each chromosome.The nCounter Human Karyotype Panel utilizes the same work flow as the nCounter Custom CNV Assay. Prior to hybridization, 600ng of genomic DNA is fragmented and denatured to yield single-stranded targets for hybridization with the Karyotype Panel CodeSet. After hybridization samples are transferred to the nCounter Prep Station where unhybridized probes are removed and probe/target complexes are aligned and immobilized in the nCounter Cartridge. Cartridges are then placed in the nCounter Digital Analyzer for data collection. Analysis of Karyotype Panel data is automated by the nCounter CNV Collector Tool provided free with the panel.
Validation of Microarray Results
Microarrays are based on the same principle as Northern blots, differential hybridization and other hybridization-based techniques. Hits from microarrays are validated for two reasons: 1) to verify the observed changes to ensure that they are reproducible in a larger number of samples, and 2) to verify the array results to ensure that they did not result from problems inherent to the array technology.
Additionally, the relatively limited dynamic range of fluorescent microarrays places limits on the technology with respect to sensitivity and specificity. Therefore, it is essential to use independent means to verify that the genes of interest are truly differentially expressed and to what extent.
SYBR Green Chemistry
SYBR Green is an intercalating dye that fluoresces upon binding to double-stranded DNA. Intercalating dyes are inexpensive and easy to use, and they can be used for any reaction because they are not sequence specific. Because they do not discriminate between specific sequences, however, they cannot be used for multiplexed analysis.
Analysis of real-time qPCR data can be either of absolute or relative levels. The majority of NASR’s analyses use relative quantitation, which is easier to measure and of primary interest to researchers examining disease states.
For absolute quantitation, an RNA standard curve of the gene of interest is required to calculate the number of copies.
For relative quantitation, comparative Ct is the most common method. The NASR performs basic analysis of individual qPCR or lower throughput experiments. The Biomedical Informatics Shared Resource analyzes high-throughput experiments including all TaqMan low density arrays using the Integromics RealTime StatMiner® software.