Improved biplex quantitative real-time polymerase chain reaction with modified primers for gene expression analysis

Stabilizing modified bases incorporated in primers allows the reduction of housekeeping gene primer concentration not possible with regular primers without sacrificing amplification efficiency. Low primer concentration allows coamplification of the most abundant housekeeping genes with very rare templates without mutual inhibition. Real-time polymerase chain reaction (PCR) coamplification of 18S ribosomal RNA with several genes of interest was used in this study with MGB Eclipse (Nanogen, San Diego, CA) hybridization probes. The results may be useful for high throughput gene expression studies as they simplify validation experiments.     

Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber

Quantitative real-time polymerase chain reaction (QRT-PCR) has become one of the most widely used methods for gene expression analysis. However, the expression profile of a target gene may be misinterpreted due to unstable expression of the reference genes under different experimental conditions. Thus, a systematic evaluation of these reference genes is necessary before experiments are performed. In this study, 10 putative reference genes were chosen for identifying expression stability using geNorm, NormFinder, and BestKeeper statistical algorithms in 12 different cucumber sample pools, including those from different plant tissues and from plants treated with hormones and abiotic stresses. EF1α and UBI-ep exhibited the most stable expression across all of the tested cucumber samples. In different tissues, in addition to expression of EF1α and UBI-ep, the expression of TUA was also stable and was considered as an appropriate reference gene. Evaluation of samples treated with different hormones revealed that TUA and UBI-ep were the most stably expressed genes. However, for abiotic stress treatments, only EF1α showed a relatively stable expression level. In conclusion, TUA, UBI-ep, and EF1α will be particularly helpful for reliable QRT-PCR data normalization in these types of samples. This study also provides guidelines for selecting different reference genes under different conditions.     

Reference gene selection for quantitative real-time polymerase chain reaction in Populus

Accurate quantification of gene expression with quantitative real-time polymerase chain reaction (qRT–PCR) relies on the choice of an appropriate reference gene. In this study, nine candidate reference genes were selected to study the expression stability for qRT–PCR normalization in adventitious rooting of Populus hardwood cuttings. geNorm, NormFinder, and BestKeeper analysis revealed that actin isoform B (ACT) was the most unstable gene across developmental stages, whereas elongation factor 1 alpha (EF1a) and 18S recombinant RNA (18S) emerged as the most appropriate reference genes for qRT–PCR analysis in this complex developmental process.     

Reference genes for quantitative reverse transcription-polymerase chain reaction expression studies in wild and cultivated peanut

Background

Molecular genetic studies on rare tumour entities, such as bone tumours, often require the use of decalcified, formalin-fixed, paraffin-embedded tissue (dFFPE) samples. Regardless of which decalcification procedure is used, this introduces a vast breakdown of DNA that precludes the possibility of further molecular genetic testing. We set out to establish a robust protocol that would overcome these intrinsic hurdles for bone tumour research.

Findings

The goal of our study was to establish a protocol, using a modified DNA isolation procedure and quality controls, to select decalcified samples suitable for array-CGH testing. Archival paraffin blocks were obtained from 9 different pathology departments throughout Europe, using different fixation, embedding and decalcification procedures, in order to preclude a bias for certain lab protocols. Isolated DNA samples were subjected to direct chemical labelling and enzymatic labelling systems and were hybridised on a high resolution oligonucleotide chip containing 44,000 reporter elements. Genomic alterations (gains and losses) were readily detected in most of the samples analysed. For example, both homozygous deletions of 0.6 Mb and high level of amplifications of 0.7 Mb were identified.

Conclusions

We established a robust protocol for molecular genetic testing of dFFPE derived DNA, irrespective of fixation, decalcification or sample type used. This approach may greatly facilitate further genetic testing on rare tumour entities where archival decalcified, formalin fixed samples are the only source.     

A quantitative multistandard reverse transcriptase-polymerase chain reaction assay of the cystic fibrosis transmembrane conductance regulator: its usefulness in studying efficiency of gene transfer

Procedures to quantify cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels have already been described but are not universally accepted, and many investigators are skeptical about quantification. To be able to accurately monitor gene therapy, we developed a quantitative multistandard RT-PCR method. This was based on the observation that the CFTR and ribosomal phosphoprotein PO (PR-PO) genes have retained important sequence homologies between rat and human species, allowing the use of rat RNA as an internal standard. A mixture of rat and human RNAs is simultaneously reverse-transcribed in one reaction tube and amplification of CFTR leads to rat and human amplificates with identical sizes which will be discriminated by restriction analysis. PR-PO is analyzed similarly and serves as a control of template loading. RT-PCR of different amounts of RNAs gave similar CFTR/PR-PO ratios, with a coefficient variation below 10%. This technique was applied to a cell line of cystic fibrosis tracheal gland serous cells (CF-KM4) incubated with a recombinant adenovirus containing the CFTR cDNA. Kinetics and dose dependency of transgene expression could be accurately quantified. This method is precise, reproducible, and very simple and could be applied to monitor gene therapy in minute amounts of tissue such as biopsies from cystic fibrosis patients.