Gene expression in the heart Quantitation of m

Gene expression in the heart Quantitation of m. RNA Methods in Cardiac Research 2016 Ulla H. Enger

Overview • RT-q. PCR is a method for quantifying RNA • RT-q. PCR – Reverse transcription (RT): Conversion of m. RNA to complementary DNA (c. DNA) – Quantitative Real-time Polymerase Chain Reaction (q. PCR): Simultaneously amplification and quantification of a DNA (or c. DNA) sequence of interest • Important to minimize and correct for experimental variations

Sample preparation RNA isolation Avoid degradation and contamination! • Snap freeze • Gloves and nuclease free consumables

RNA concentration and quality RNA concentration and purity measured by Nanodrop

RNA quality Bioanalyzer: • Estimate RNA degradation • RIN: RNA integrity number • Ideally 10

c. DNA synthesis Reverse transcription (RT) • Conversion of m. RNA to c. DNA (complementary DNA) by the enzyme reverse transcriptase and primers • RT primer strategies – – – Gene specific Oligo (d. T) Random hexamers

c. DNA synthesis Reverse transcription (RT) Ref: http: //pathmicro. med. sc. edu/pcr/pp-pcr 1. gif

Polymerase Chain Reaction (PCR) • In vitro method for amplification of a specific DNA sequence • Traditional PCR End point (gel electrophoresis and image analysis) • q. PCR The amount of PCR product in mesured for each cycle. Allows amplification and quantitation of DNA

q. PCR Primer design Design your own or use pre designed primer/probes? Key factors: • Primer length • Primer specificity • Melting temperature • Base distrubution • Sencondary structure

q. PCR chemistry Ref: http: //www 3. appliedbiosystems. com/cms/groups/portal/documents/web_content/cms_052221. png

q. PCR Platau phase Exponential phase Fluoresence Ct value Threshold Background fluoresence PCR cycle Emitted fluorescense reflects the amount of amplified DNA

q. PCR Standard curve: • Optimization of q. PCR reaction • Calculation of RNA abundance • Effectivity: E=10(-1/slope)-1 • Slope -3, 32 = 100% effectivity • Slope between -3, 58 and -3, 10 is OK

q. PCR quantitation wt. AB wt. SHAB Relative m. RNA abundance Methods for quantitation: • Relative quantitation; relate to relative standard curve or reference gene • Absolute quantitation; relate to standard curve made from known standards 18 16 14 12 10 8 6 4 2 0 Myh 7 wt. AB wt. SHAB Relative m. RNA abundance wt SHAB 2 1. 8 1. 6 1. 4 1. 2 1 0. 8 0. 6 0. 4 0. 2 0 * wt AB Gapdh wt SHAB wt AB

q. PCR Normalization • Correction of variable input of template, quality and/or RT effectivity • There are no ‘perfect’ reference genes • Reference genes must be expessed constant in your material • Single reference gen or a set of genes with minimal variation? Brattelid et al, 2010

Digital PCR (d. PCR) • A novel method for precise quantification of nucleic acids • Counts the total number of individual target molecules in a digital format • Absolute quatitation, higher senstitivity and lower sample requirment • In general, more labour intensive method

Digital droplet PCR (dd. PCR) Run PCR Data anaysis Make droplets Read droplets

References: • • S. A. Bustin et al. , The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments, Clin. Chem. 55 (2009), pp. 611– 622. Nolan, T, Hands, R. E and Bustin, S. A, Quantification of m. RNA using the real-time reverse transcriptation polymerase chain reaction, Nature Protocols 1, - 1559 - 1582 (2006) Bustin, S. A, Nolan, T. , Pitfalls of Quatitative Real-Time Reverse Transcription Polymerase Chain Reaction, Journal of Biomolecular Techniques, 2004, 15: 155 -166 Fleige S, Pfaffl MW. RNA integrity and the effect on the real-time q. RT-PCR performance. Mol Aspects Med. 2006 Apr-Jun; 27(2 -3): 126 -39. Review.