Lecture 3 - Transcriptomics (PDF)

Lecture 3 SEPTEMBER 10, 2024 Transcriptomics  Real-Time PCR (qPCR)  Microarray  Next generation sequencing PCR  Amplification of DNA  Can be used for detection, cloning and sequencing of specified regions  Uses one or more primers https://www.genome.gov/genetics-glo...

Lecture 3 SEPTEMBER 10, 2024 Transcriptomics  Real-Time PCR (qPCR)  Microarray  Next generation sequencing PCR  Amplification of DNA  Can be used for detection, cloning and sequencing of specified regions  Uses one or more primers https://www.genome.gov/genetics-glossary/Polymerase-Chain-Reaction Quantitative PCR 1. Reverse Transcription 2. Amplify 3. Capture fluorescence 4. Repeat Steps 2 and 3 for 29 more rounds 5. Determine Double Delta-CT 1. Gene of Interest Experimental (TE) 2. Gene of Interest Control (TC) 3. House Keeping Gene Experimental (HE) 4. House Keeping Gene Control (HC) qPCR  Delta-CT  TE-HE (ΔCTE)  TC-HC (ΔCTC)  Double Delta-CT  ΔCTE – ΔCTEC= ΔΔCT  Calculate Fold Change  2-ΔΔCT  Microarrays https://www.google.com/url?sa=i&url=https%3A%2F%2Fold-ib.bioninja.com.au%2Foptions%2Funtitled%2Fb4-medicine%2Fdna-microarrays.html&psig= AOvVaw0nspQ4_Gkosoy0Vh5CxYaE&ust=1726064964795000&source=images&cd=vfe&opi=89978449&ved=0CBQQjRxqFwoTCOjfopvLuIgDFQAAAAAdAAAAABAE Next Generation Sequencing  Total RNA  Poly-A Capture  Specific Probes Why do transcriptomic experiments?  General gene discovery  Compare experimental treatments  Drug treatments  Knockout/Knockin studies  Toxicology  Developmental Stages In Class Work  Split up into groups  Each group design an experiment to test changes in gene expression  State assumptions going into the experiment  List expected results  List potential pitfalls