Category
Poster - Basic
Description
Reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive technique commonly used to characterize gene expression in cells and tissues. While a standard PCR requires hours to complete, a modified “fast” PCR takes ~30 minutes which could allow for advanced integration into undergraduate laboratory courses. Therefore, studies were performed to flashlight putative “fast” PCR efficacy by detecting anti-Müllerian hormone (AMH). AMH is a peptide hormone regulating sex development. In adult male mammals, AMH supports sperm production. It was hypothesized that AMH gene expression in testes could be detected by RT- “fast” PCR, an accelerated protocol, to meet teaching-lab timeframes. Total RNA was isolated from fresh frozen mouse testes using a rapid spin column-based method and analyzed by spectrophotometry. RT was performed using a commercial kit to generate cDNA templates. PCR was employed at “standard” (95°C for 3 minutes; 40 cycles of 95°C for 30 seconds, 55°C for 40 seconds, 72°C for 30 seconds; 72°C for 5 minutes) and “fast” (98°C for 40 seconds; 30 cycles of 92°C for 2 seconds, 60°C for 2 seconds; 72°C for 5 minutes) pace to amplify AMH and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; housekeeping gene). PCR products were analyzed by gel electrophoresis. Experiments were replicated 7 times. Rapid isolation delivered RNA of quality >2.10 (OD260/280) and quantity 491.4 ug/ml. Both AMH and GAPDH were detected by standard PCR. While GAPDH was detected by “fast” PCR, results for AMH were inconclusive. Bridging the undergraduate-graduate dichotomy, an RT-“fast” PCR methodology proved conducive to GAPDH presentation. Future work could further modify the protocol for AMH detection by increasing cDNA concentration, attenuating annealing/extension temperature, and increasing cycle number, with a goal of integrating this technique into undergraduate curricula. The efficiency gains could afford Liberty students pedagogy often suspended until post-graduate endeavors, inculcating competitive advantage generative of downstream academic and industrial success.
Anti-Müllerian Hormone Gene Expression in Mouse Testes Elucidated via Reverse Transcription Polymerase Chain Reaction
Poster - Basic
Reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive technique commonly used to characterize gene expression in cells and tissues. While a standard PCR requires hours to complete, a modified “fast” PCR takes ~30 minutes which could allow for advanced integration into undergraduate laboratory courses. Therefore, studies were performed to flashlight putative “fast” PCR efficacy by detecting anti-Müllerian hormone (AMH). AMH is a peptide hormone regulating sex development. In adult male mammals, AMH supports sperm production. It was hypothesized that AMH gene expression in testes could be detected by RT- “fast” PCR, an accelerated protocol, to meet teaching-lab timeframes. Total RNA was isolated from fresh frozen mouse testes using a rapid spin column-based method and analyzed by spectrophotometry. RT was performed using a commercial kit to generate cDNA templates. PCR was employed at “standard” (95°C for 3 minutes; 40 cycles of 95°C for 30 seconds, 55°C for 40 seconds, 72°C for 30 seconds; 72°C for 5 minutes) and “fast” (98°C for 40 seconds; 30 cycles of 92°C for 2 seconds, 60°C for 2 seconds; 72°C for 5 minutes) pace to amplify AMH and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; housekeeping gene). PCR products were analyzed by gel electrophoresis. Experiments were replicated 7 times. Rapid isolation delivered RNA of quality >2.10 (OD260/280) and quantity 491.4 ug/ml. Both AMH and GAPDH were detected by standard PCR. While GAPDH was detected by “fast” PCR, results for AMH were inconclusive. Bridging the undergraduate-graduate dichotomy, an RT-“fast” PCR methodology proved conducive to GAPDH presentation. Future work could further modify the protocol for AMH detection by increasing cDNA concentration, attenuating annealing/extension temperature, and increasing cycle number, with a goal of integrating this technique into undergraduate curricula. The efficiency gains could afford Liberty students pedagogy often suspended until post-graduate endeavors, inculcating competitive advantage generative of downstream academic and industrial success.
Comments
Undergraduate