Biochemical and transcriptomic analyses reveal different metabolite biosynthesis profiles among three color and developmental stages in ‘Anji Baicha’ (Camellia sinensis)

Primers used for quantitative reverse transcription polymerase chain reaction analyses. (XLSX 11 kb

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and its usefulness in soil microbial ecological studies - A review

The reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a highly specific polymerase chain reaction (PCR) method that allows one to detect very low transcription levels of functional gene(s) in soil. RT-qPCR helps us to know the active members of the microbial community, and their activities can be linked with other ecological processes in soil. If after the extraction of RNA from soil, the mRNA is converted to cDNA which is then sequenced, one would analyze directly the active members of the microbial community.Key words: Complementary DNA (cDNA), messenger RNA (mRNA), reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), soil microbial study, microbial community

Virus-transformed pre-B cells show ordered activation but not inactivation of immunoglobulin gene rearrangement and transcription

Virus-transformed pre-B cells undergo ordered immunoglobulin (Ig) gene rearrangements during culture. We devised a series of highly sensitive polymerase chain reaction assays for Ig gene rearrangement and unrearranged Ig gene segment transcription to study both the possible relationship between these processes in cultured pre-B cells and the role played by heavy (H) chain (mu) protein in regulating gene rearrangement. Our analysis of pre-B cell cultures representing various stages of maturity revealed that transcription of each germline Ig locus precedes or is coincident with its rearrangement. Cell lines containing one functional rearranged H chain allele, however, continue to transcribe and to rearrange the allelic, unrearranged H chain locus. These cell lines appear to initiate but not terminate rearrangement events and therefore provide information about the requirements for activating rearrangement but not about allelic exclusion mechanisms

Detection of potato leafroll virus isolated from potato fields in Tehran province in aphids by immunocapture reverse transcription polymerase chain reaction

The surveys conducted during 2006 and 2007 revealed the infection of the virus in potato fields in Tehran province. Due to the important roll of aphids in transmission of the virus, immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR) and reverse transcription polymerase chain reaction (RT-PCR) was developed using potato leafroll virus (PLRV) specific antibodies and specific primer pair (20 mer) located in the virus capsid gene to detect the virus in aphids. A 336-bp PCR product was detected from aphids (Myzus persicae) which had been fed on PLRV-infected plants. The PCR band was specific to PLRV as determined in PLRV-infected plants. This inquiry shows that this method is applicable to assess viroliferous nature of aphids in yellow -pan traps during season

Identification of genes differentially expressed in Jining Grey and Liaoning Cashmere goats ovaries

To search for genes controlling high prolificacy of Chinese indigenous goats, differential display reverse transcription-polymerase chain reaction (DDRT-PCR) was used to screen differentially expressed cDNA bands in the sexually matured ovaries of 3-year-old prolific Jining Grey goats and monotocous Liaoning Cashmere goats with 24 combinations of three anchored primers and eight arbitrary primers. 22 expressed sequence tags (ESTs) were proved to be the positive bands by Northern hybridization. They comprised 10 known ESTs and 12 ESTs without homologous sequences in the GenBank. These results indicate that several genes such as GATA-4, metallothionein-like protein, CAT genes and unknown ESTs (CV983340 and CV983341) were expressed only in Jining Grey goats.Keywords: Differential display reverse transcription-polymerase chain reaction, goat, ovary, prolificacyAfrican Journal of Biotechnology Vol. 12(27), pp. 4408-441

Analysis of RT-qPCR Data

We give a brief overview of the necessary steps in the analysis of real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) data. We cover determination of amplification efficiency, background correction, normalization, quality control, and statistical analysis

Dengue-1 Virus Isolation during First Dengue Fever Outbreak on Easter Island, Chile

Dengue virus was detected for the first time in Chile, in an outbreak of dengue fever on Easter Island. The virus was isolated in tissue culture and characterized by reverse transcription–polymerase chain reaction as being dengue type 1

Molecular diagnosis of peste des petits ruminants virus (PPR) in goats and sheep populations

Peste des petits ruminants (PPR) is an economically important viral disease of goats and sheep. The disease is confused clinically with other infections such as the mild strain of rinderpest in small ruminants. Effective control measures for PPR need that a proper and rapid diagnostic technique of disease. Therefore, the use of reverse transcriptase-polymerase chain reaction (RT-PCR) to detect suspected field samples collected from diseased goats and sheep in Dammam city, Kingdom of Saudia Arabia (KSA) has helped to give an effective diagnosis that was needed to control measure of the spread of the disease. This assay is based on the rapid purification of RNA on glass beads followed by the reverse transcription-polymerase chain reaction (RT-PCR). The primers (NP3/NP4) were used to amplify specifically a fragment of about 350 bp, that technique has a more specific and sensitive method for rapid diagnosis of disease.Key words: Peste des petits ruminants virus; reverse transcriptase polymerase chain reaction; diagnosis; goats; shee

Accurate RT-qPCR gene expression analysis on cell culture lysates

Gene expression quantification on cultured cells using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) typically involves an RNA purification step that limits sample processing throughput and precludes parallel analysis of large numbers of samples. An approach in which cDNA synthesis is carried out on crude cell lysates instead of on purified RNA samples can offer a fast and straightforward alternative. Here, we evaluate such an approach, benchmarking Ambion's Cells-to-CT kit with the classic workflow of RNA purification and cDNA synthesis, and demonstrate its good accuracy and superior sensitivity