Sample Preparation and Reaction Set-up

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1 Sample Preparation and Reaction Set-up

2 Important aspects of sample preparation and reaction setup Sampling Sample storage RNA / DNA Extraction and quality RNA / cdna / DNA storage Primer and probes formulation and storage RT and PCR setup Controls 2

3 Sampling Blood Cell culture Tissue Paraffin embedded tissue Biopsy with limited sample Others what do you use? 3

4 RNA Workflow RNA stabilization conditions that prevent RNA degradation (inactivates RNAses) and stabilize gene expression profiles 4

5 Sample Handling before Lysis Effect on Gene Expression in Blood Samples 5 Note: Tanner et al., Clin.Lab.Haem 2002, 24,

6 RNA Workflow 6

7 RNA / DNA extraction Two main goals: Release nucleic acids with high purity and high yield Remove PCR inhibitors 7

8 RNA / DNA Extraction Methods What do YOU use? Chaotropic reagents (Phenol / Chloroform, TRI reagents, etc) Spin columns Silica Based membranes Magnetic beads Boil & Spin Note: See Sambrook et al. (CSH editions) for generic protocols 8

9 RNA Yield-General Guidelines The Mouse Tissue RNase Hierarchy Thus, basal RNase activity from Mouse Pancreas is 17x that of Spleen, and 181,000x that of Brain 9

10 RNA / DNA quality UV Spectrophotometry Purity: UV Analysis OD260/280 RNA: OD260/280 ratio (OD µg/ml) DNA: OD260/280 ratio (OD µg/ml) Lowered ratios mean protein contamination Increased 410 nm means heme contamination Increased 230 nm means residual organics 10

11 RNA / DNA quality UV Spectrophotometry + _ widely used, simple uses common equipment less sensitive than other methods DNA, RNA, free nucleotides and anything that absorbs at 260nm will interfere sometime poor equipment 11

12 UV analysis OD 260/ A260/280 Ratio versus % Protein in Raji RNA A260/280 Ratio A260/280 Ratio % Protein w/w (BSA) Note: OD 260/ could mean 60-80% of protein in RNA samples!! See Appendix: A. Tanner et al., Clin.Lab.Haem 2002, 24,

13 + _ RNA / DNA quality Fluorescent Dye The fluorescence of a sample containing this dye is measured in a fluorimeter and compared to a standard curve More sensitive (x100) than UV spectrophotometry Less sensitive to protein and free nucleotide interference DNA or RNA contamination can cause inaccurate quantitation Requires a fluorimeter 13

14 DNA Quality /Quantity Agarose Gel Electrophoresis Reveals the presence of contaminating DNAs and RNAs, but not proteins Purified DNA runs as a single band Estimate DNA amount using ladder with fragments of known concentration (eg. Low DNA Mass Ladder) 14

15 RNA Quality /Quantity Denaturing agarose Gel Electrophoresis + RNA will run by size. The 28S and 18S rrna bands are visualized by ethidium bromide staining. Widely used, simple _ uses common equipment Considerably less sensitive than other methods Subjective because appearance of rrna bands is affected by electrophoresis conditions, amount of RNA loaded, and saturation of ethidium bromide fluorescence 15

16 RNA Quality /Quantity Combination of microfluidics, capillary electrophoresis, and fluorescent dyes An RNA-specific dye is added to the sample and quantitation is accomplished by comparing fluorescence of the sample to that of a standard (Agilent 2100 bionalayzer). + Requires small amount of RNA Quantitate RNA and assess RNA integrity (RIN) Quantitation not affected by common contaminants _ Instrument not available in many labs 16

17 Template purity Protein contamination Unstable RNA/DNA because of Nucleases Loss of RNA/DNA during storage over time No reproducible assay performance PCR inhibitors Loss of sensitivity 17

18 PCR inhibitors Sample related: Heparin Hemoglobin > 0,15 mg/ml > 1 mg/ml Melanin, humic acids, chlorophyll, polysaccharides Extraction related: SDS > 0,01% (w/v) Phenol > 0,2% (v/v) Ethanol > 1% Sod. acetate > 5 mm 18 PCR additives: DTT > 1 mm DMSO > 5 % Mercaptoethanol

19 Important aspects of sample preparation and reaction setup Sampling Sample storage RNA / DNA Extraction and quality RNA / cdna / DNA storage Primer and probes formulation and storage RT and PCR setup Controls 19

20 RNA and DNA storage RNA Optimally at 80 C for 1-2 years RNase free H2O mm EDTA TE low buffer (1 mm Tris, 0.01 mm EDTA) Store undiluted Not in ethanol DNA At 20 C in TE low buffer or ethanol Store undiluted 20

21 Important aspects of sample preparation and reaction setup Sampling Sample storage RNA / DNA Extraction and quality RNA / cdna / DNA storage Primer and probes formulation and storage RT and PCR setup Controls 21

22 Creating probe stock solutions TAMRA dye-labeled probes Arrive lyophilized, dilute in 1 mm Tris HCl (ph 8), 0.01 mm EDTA Stock concentration e.g. 10 µm MGB probes Arrive diluted (100 µm), at room temperature (stable for days) dilute in 1 mm Tris HCl (ph 8), 0.01 mm EDTA Stock concentrations e.g. 10 µm 22

23 Creating primer and probe stock solutions Primers Arrive lyophilized Dilute in 1 mm Tris-HCl (ph 8), 0.01 mm EDTA Stock concentrations e.g. 10 µm in TE buffer TaqMan assays Primer-probe mixtures Differently concentrated, can directly be used as stock solution. 23

24 Primer and probe storage Aliquot stock solutions Reduced number of freezing and thawing processes Potential contamination would affect only one aliquot and not entire stock solution Aliquot volume based on throughput Store aliquots at -20 C 24

25 Important aspects of sample preparation and reaction setup Sampling Sample storage RNA / DNA Extraction and quality RNA / cdna / DNA storage Primer and probes formulation and storage RT and PCR setup Controls 25

26 Real-time PCR plate setup Analysis of genomic DNA Gene Expression Studies Analysis of viral RNA 26

27 Setup for genomic DNA DNA PCR Master Mixes or Core Reagents Kit or Microorganism Detection Kit Applications SNP genotyping Copy Number Variation Drug Metabolizing Enzymes Microrganism Detection 27

28 Setup for viral RNA: One-step RT-PCR protocols Viral RNA One-Step RT-PCR Master Mix or Core Reagents Kit or Microorganismes Detection Kit Applications Viral detection and quantitation Gene expression 28

29 Setup for gene expression: Two-Step Protocol Total RNA Reverse Transcription Reagents or RT-PCR kit cdna Pool PCR Master Mixes or Core Reagents Kit Applications Gene Expression 29

30 Reverse Transcription (RT) Aspects of cdna Synthesis One-step versus two-step RT-PCR? RT primer? random hexamers oligo dt gene-specific primers Choice of enzyme: this choice is application- and templatedependant Applications: mirna, Gene Expression or viral studies Template: if secondary structure present, choose an enzyme capable of working at higher temperatures Efficiency of RT step? Tip: Run RT at highest possible temperature 30

31 Reverse Transcription (RT) Aspects of cdna Synthesis RT directly after extraction or batch collection? Tip: Perform steps with all samples in parallel, if possible Reproducibility? Once the conditions choosen(rna concentration & quality, temperature, enzyme) the same setup time is an important aspect for reproducibility Low template concentrations / low copy number Tip: Use carrier RNA (e.g. Yeast t-rna) to avoid adsorbtion to plastic 31

32 How much template per well? DNA 100 pg 1 µg SNP Genotyping 5-20 ng cdna Gene Expression Low Density Array 10 pg 100 ng 1-50 ng ng per filling port RNA 100 pg 1 µg mrna 10 pg 100 ng mirna 1-10 ng (20ng total RNA if using the 48-plex RT protocol) 32

33 Is it Possible to Analyse low Expressed genes from Minute RNA Samples? Formalin fixed paraffin-embedded tissues Laser capture micro dissections Needle biopsies Flash frozen tissue samples Single Cells 33

34 Preamplification Concept Conventional gene expression analysis Reverse transcribe RNA to cdna Perform real-time PCR with TaqMan Assays RNA cdna Gene expression analysis with preamplification Reverse transcribe RNA to cdna Preamplify cdna using pool of TaqMan Assays Perform real-time PCR with same TaqMan Assays RNA cdna 34

35 Pre-amplification workflow PreAmp Master Mix 10 or 14 cycles of PCR TaqMan Gene Expression Master Mix cdna Load Samples onto SDS platform Pooled Assays (primers needed for PCR) 1:5 or 1:20 Dilution (1X TE) Temperature Time Parameter 95C 10 minutes Hold 95C 15 seconds 60C 4 minutes x10 or 14 cycles TaqMan Gene Expression Assay 35

36 An example of experimental testing Input cdna can range from 1-250ng Experimental Design Preamp: 100-plex assay pool 10 cycle preamp UHR cdna using 1 ng input Single-plex: Gene Expression Master Mix Assayed 96 of the 100 targets 1:20 dilution of PreAmp products 36

37 100-Plex preamplification performance: 1ng cdna input 1ng cdna PreAmp vs Unamplified cdna Ave Ct: Preamp Ave Ct: cdna Slope R Data generated by comparing pre-amplified UHR cdna to unamplified UHR cdna. Preamplification was performed at 100plex, with 1ng of cdna input. 96 of the 100 TaqMan gene expression targets were assayed in quadruplicate. For comparison, the same 96 targets were assayed using 0.3ng/uL of unamplified UHR cdna. Average Ct values are compared for the pre-amplified and unamplified cdna templates. There is minimal to no bias introduced during the pre-amplification step. 37

38 Preamplification performance: various plexity levels Comparison of 13 Assays: 16 plex, 48 plex & 96 plex Preamplifications ddct Value Assay 16 Plex 48 Plex 96 Plex Three pre-amplification assay pools were generated by combining 16, 48 and 96 TaqMan Gene Expression assays. Thirteen of the assays were common to all 3 pools. Preamplification reactions were performed using all 3 pools and 25ng s UHR cdna input for 10 cycles of PCR. Preamplification products were used as the templates for single-plex TaqMan Gene Expression reactions (run in quadruplicate). Ct values were determined by normalizing all data to the CDKN1B assay. Ct values were then determined by taking the difference between the cdna Ct and the pre-amplified cdna Ct values. All 13 assays have very similar performance, regardless if they undergo a 16-plex, 48-plex or 96-plex pre-amplification reaction. 38

39 Real-time PCR plate setup Area 1 PCR mix preparation Distribute PCR mix Use of of pipette set 1 Area 2 Add template Close plate Use of of pipette set 2 Area 3 Run PCR Trash plate 39

40 Setup recommendations Use pipette tips with filters Calibrate your pipettes on a regular basis Aliquot reagents Check water quality ph, salt, fluorescent contaminations AMBION Water for MB Monitor and prevents environmental RNAse contamination (use RNaseZap ) Prevents DNA and RNA contamination in the lab (use DNAZap ) 40

41 What about replicates and controls? Number of PCR replicates required is given by the precision you demand Run no template controls (NTC s) for each assay used on the plate RT minus controls for the detection of genomic DNA contamination 41

42 nm, pmol etc... How many µl of a 10 µm primer stock solution do we have to add to a 50 µl PCR reaction to get a final concentration of 900 nm? 42

43 nm, pmol etc µm means 10 µmoles/l = 10 pmoles/µl 900 nm final means 900 nmoles/l = 900 pmoles/ml = 45 pmoles/50 µl µl 43

44 Master Mix preparation Excel sheets are on your Training CD 44

45 Legal Statements For Research Use Only. Not for use in diagnostic procedures. NOTICE TO PURCHASER: PLEASE REFER TO THE USER'S GUIDE OR PACKAGE INSERT OF THE PRODUCTS NAMED HEREIN FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION. Applera, Applied Biosystems, AB (Design) are registered trademarks and TAMRA is a trademark of Applera Corporation or its subsidiaries in the US and/or certain other countries. TaqMan is a registered trademark of Roche Molecular Systems, Inc. SYBR green is registered trademark of Molecular Probes, Inc. All other trademarks are the sole property of their respective owners Applied Biosystems. All rights reserved. 45

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