Selection of a stable reference gene for relative copy number profiling of porcine chromosomal genes using SYBR green qPCR

Sahoo, Nihar Ranjan; Pruthviraj, D. R.; Yadav, Vandana; Santhosh, Ameya; Maurya, Rajveer; Gaur, Gyanendra Kumar; Kumar, Pushpendra; Ravikumar, G.

doi:10.1080/10495398.2022.2092742

Cited by 2 publications

(4 citation statements)

References 21 publications

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“…20 The higher the starting copy number, the lower its CT value, and vice versa. 21 Fold differences for each transcript expression in Control versus Treated groups are defined by 2 ÀΔΔCt (Equations 1 and 2).…”

Section: Results Of Experimentsmentioning

confidence: 99%

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Development of an integrated and project‐based laboratory course in upper‐level biochemistry and molecular biology

Liu,

Tu,

et al. 2024

Biochem Molecular Bio Educ

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An integrated and projected‐based laboratory course was described, integrating interconnected knowledge points and biochemistry and molecular biology techniques on a research project‐based system. The program, which served as an essential extension of theoretical courses to practice, was conducted with a sophomore of basic medical science who had completed the course in medical biochemistry and molecular biology. This course engaged students in learning “genetic manipulation” and “recombinant DNA technology” to understand the target gene's role in disease mechanics, thus altering evaluation and treatment for clinical disease. Students could master applied and advanced techniques, such as cell culture, transfection, inducing exogenous fusion protein expression, purifying protein and its concentration assay, quantitative polymerase chain reaction, and western bot analysis. This laboratory exercise links laboratory practices with the methods of current basic research. Students need to complete the experimental design report and laboratory report, which could be advantageous for improving their ability to write lab summaries and scientific papers in the future. The reliability and validity analyses were conducted on the questionnaire, and we examined students' satisfaction with the course and their gains from the course. The student feedback was generally positive, indicating that the exercise helped consolidate theoretical knowledge, increase scientific research enthusiasm, and provide a powerful tool to be a better person and make informed decisions.

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Section: Results Of Experimentsmentioning

confidence: 99%

“…The CT value represents each PCR reaction's cycle when the fluorescent signal reaches the set threshold 20 . The higher the starting copy number, the lower its CT value, and vice versa 21 . Fold differences for each transcript expression in Control versus Treated groups are defined by 2 −∆∆Ct (Equations 1 and 2).…”

Section: Resultsmentioning

confidence: 99%

Development of an integrated and project‐based laboratory course in upper‐level biochemistry and molecular biology

Liu,

Tu,

et al. 2024

Biochem Molecular Bio Educ

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“…Out of the 11 pairs of primers tested (Table 1), only six pairs amplified successfully in porcine male DNA producing specific bands. Of these six successful primer pairs, five genes were amplified from sex chromosomes (Table 2) and IGF1R from chromosome number 1 (Autosome), which was selected from previous reports (Sahoo et al, 2022). The sex chromosomal primer pairs were evaluated for sex chromosome‐specific amplification using cyto‐screened pig DNA of either sex (Table 2) which revealed male‐specific amplification in two genes (EIF1AY and SMCY).…”

Section: Resultsmentioning

confidence: 99%

“…Our group previously reported differential Y amplification and the copy number profiling of X‐ and Y‐specific gene fragments in crossbred pigs with numerically normal karyotype (Yadav et al, 2020) using a random autosomal gene fragment as reference gene. Furthermore, we evaluated the copy number stability for seven genes (FSHB, IL4, IGF1R, TCF24, BRMS1L, ARMC1 and SRSF4) selected the most stable reference gene for copy number analysis of chromosomal genes based on the chromosomal location, reports of single copy and lack of involvement in structural chromosomal abnormalities (Sahoo et al, 2022). The present study aimed to design a qPCR‐based method for porcine semen sex ratio determination which estimates the proportionate contribution of X‐ and Y‐bearing spermatozoa with an autosomal reference gene.…”

Section: Introductionmentioning

confidence: 99%

Genetic group and swim‐up processing affect SYBR Green real‐time PCR‐based porcine semen sex ratio in Landrace and its crossbred boars

Santhosh

Sahoo

Yadav

et al. 2023

Reprod Domestic Animals

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Determination of factors affecting sex ratio is important while considering application of sex ratio enrichment approach. Present study aimed to design a SYBR Green qPCR‐based method for measurement of primary sex ratio and to evaluate different factors (genetic group, sire, spermiogenic cycle and processing layer) affecting boar sperm sex ratio. The qPCR was based on relative copy number analysis of sex chromosome‐specific single copy gene fragments with an autosomal gene as reference and was evaluated using DNA dilution series from pigs with numerically normal karyotype. The sex ratio was estimated from genomic DNA samples isolated from boar semen collected from different genetic groups at different time points and different processing layers. The X chromosome frequencies of semen samples revealed significant effect of genetic group. However, significant variation was observed neither within same genetic group nor between ejaculates of different spermatogenic cycles. Among the processing techniques studied, swim‐up technique produced a significant X sperm enrichment in comparison to control whereas, Percoll density gradient failed to show any significant difference among layers. The lower layer in swim‐up technique was found to contain higher proportion of X sperms. The designed qPCR is found to be an easy, less time‐consuming method and does not require high end laboratory facilities or the specialized expertise. The lower layer of swim‐up processing has a scope for X sperm enrichment in boar semen with proper validation.

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Selection of a stable reference gene for relative copy number profiling of porcine chromosomal genes using SYBR green qPCR

Cited by 2 publications

References 21 publications

Development of an integrated and project‐based laboratory course in upper‐level biochemistry and molecular biology

Development of an integrated and project‐based laboratory course in upper‐level biochemistry and molecular biology

Genetic group and swim‐up processing affect SYBR Green real‐time PCR‐based porcine semen sex ratio in Landrace and its crossbred boars

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