Understanding Melt Curves for Improved SYBR® Green Assay Analysis and Troubleshooting

qPCR assays using intercalating dyes, such as SYBR® Green dye, are an economical and effective tool for measuring gene expression. To interpret intercalating dye assays, users need to know how to analyze melt curves, and understand the benefits and limitations of melt curve analysis. In this presentation, Nick Downey, PhD, covers melt curve basics and shares examples of multiple peaks due to suboptimal sample prep, primer dimers, and asymmetric GC content of amplicons. He demonstrates troubleshooting strategies. Experienced and novice users will benefit from an overview of uMeltSM software, developed by the Wittwer lab at the University of Utah, that can predict the melt profile of your assay before you run your experiment.

1. Understanding Melt Curves for Improved SYBR ® Assay Analysis and Troubleshooting April 2, 2015 Dr Nick Downey, Applications Scientist

2. Outline Review of intercalating dye–based qPCR Theory of melt curves How melt curves can help diagnose problems Use of Umelt SM software to help with data interpretation Troubleshooting SYBR ® dye–based experiments Steps to successful qPCR design

3. qPCR —Intercalating Dye vs. Probe-Based Primers Only For use with intercalating dyes such as SYBR ® Green Primers and Probe For use in the 5’ nuclease assay

4. Intercalating Dye Assays vs . 5′ Nuclease Assays Intercalating Dye Assays Inexpensive Non-specific PCR products and primer dimers will generate fluorescent signal Requires melting point curve determination Cannot multiplex Cannot be used for single-tube genotyping of 2 alleles 5′ Nuclease Assays 3 rd sequence in assay (the probe) adds specificity Specific amplification for rare transcript or pathogen detection Does not require post-run analysis such as melt curves Can multiplex Can be used for single-tube genotyping of 2 alleles

5. SYBR ® Green Dye Asymmetrical cyanine dye Intercalating dyes fluoresce only when bound to DNA Most only bind efficiently to double-stranded DNA Similar cyanine dyes SYBR ® Green II SYBR Gold PicoGreen ® DNA–dye complex: Absorbs blue light ( λmax = 497 nm) Emits green light ( λmax = 520 nm) Developed to quantify template (RNA and DNA) Preferentially binds to double-stranded DNA Lower performance with single-stranded DNA and RNA

6. Why Run Melt/Disassociation Curves W hen U sing I ntercalating Dyes SYBR ® Green dye will detect any double-stranded DNA, including: primer dimers contaminating DNA PCR product due to mis -annealed primers By viewing a dissociation/melt curve, you ensure that the desired amplicon was detected

7. Theory of Melt Curves Temperature F luorescence As the temperature is increased the DNA starts to denature

8. The Initial F luorescence D ata is Manipulated to Produce a Q uick R ead P lot

9. How Does a Melt Curve Help Data Analysis? SYBR ® Green assays detect any DNA; hence, the melt curve can indicate potential issues, such as: gDNA contamination in an RNA sample Primer-dimers affecting the assay Splice variants (if there is extra sequence between primers)

10. Problem: Small Amount of gDNA in cDNA Sample Assay targeting TCAF1  (TRPM8 channel-associated factor 1) produces a single peak No RT control also produces a single peak Sample Ladder – RT NTC

11. Problem: Small Amount of gDNA in cDNA Sample Assay targeting TCAF1  (TRPM8 channel-associated factor 1) produces a single peak No RT control is necessary for diagnosing genomic DNA contamination. No RT control also produces a single peak Sample Ladder – RT NTC

12. Problem: Large Amount of Contaminating gDNA Sample Results No Reverse Transcription Assay across intron of BAIAP3   (BAI1-associated protein 3) – RT Sample Ladder NTC

13. Problem: Large Amount of Contaminating gDNA Sample Results No Reverse Transcription Gel analysis confirms genomic DNA amplification Assay across intron of BAIAP3   (BAI1-associated protein 3) – RT Sample Ladder NTC

14. Solution: Treat RNA with More DNase Original prep of RNA used for BAIAP3  (BAI1-associated protein 3 ) amplification

15. Solution: Treat RNA with More DNase RNA for BAIAP3 amplification retreated with DNase

16. Melt Curves S how R emoval of Off-Target Amplicons RNA retreated with DNase (BAIAP3 amplification) Original RNA sample (BAIAP3 amplification)

17. Not All Primer Dimers are a Problem for an Assay Assay designed against PPIA, within a single exon NTC shows multiple peaks, raising concern about primer-dimers CE analysis indicates no problem from primer dimers – RT Sample Ladder NTC

18. Problem: Assay Designed A cross a Small Intron Low DNase High DNase gDNA High DNase treatment does not resolve the issue Possible solution: Probe-based assay across exon junction Low DNase High DNase Low DNase –RT High DNase –RT

19. Wittwer Lab is Interested in Understanding M elt C urves Designed a series of amplicons spanning exons of cystic fibrosis transmembrane receptor (CFTR) Tested each one for melt characteristics and gel mobility Developed a model for melting of amplicon DNA

20. Extra Peaks in Melt Curves Do Not Always Indicate a Problem A mplicon from exon 17b of CFTR A mplicon from exon 7 of CFTR

21. Agarose Gel E lectrophoresis is Useful for Confirming Melt Curve Data 100 bp 200 bp A B R eplicates of the amplification of CFTR exon 17b R eplicates of the amplification of CFTR exon 7 Gel electrophoresis is the best method for analyzing PCR products, but is very labor- and time-consuming.

22. DNA Melting Is Not Always Biphasic G-C-G-C-G-C-G-C-G-C-G-A-T-A-T-T-T-A-A-T-A-T-A C-G-C-G-G-C-G-C-G-C-G-T-A-T-A-A-A-T-T-A-T-A-T G-C-G-C-G-C-G-C-G-C-G-A-T-A-T-T-T-A-A-T-A-T-A C-G-C-G-G-C-G-C-G-C-G-T-A-T-A-A-A-T-T-A-T-A-T | | | | | | | | | | | | | | | | | | | | | | | -A-T-A-T-T-T-A-A-T-A-T-A G-C-G-C-G-C-G-C-G-C-G -T-A-T-A-A-A-T-T-A-T-A-T C-G-C-G-G-C-G-C-G-C-G | | | | | | | | | | | Assumed event Possible event

23. A Model for Explaining the CFTR Exon 7 Double Peak

24. Best Methods for Assessing SYBR ® Green Melt C urves Gold standard: gel electrophoresis Alternative: predict if melt occurs with more than one phase

25. uMelt SM Software Helps to Predict Melting of a PCR Product uMelt SM predicts melt behavior of PCR products: https:// www.dna.utah.edu/umelt/um.php Developed by Wittwer lab

26. uMelt SM Software Predicts Melting of CFTR Exon 7 A mplicon Different prediction models are available You can further manipulate conditions

27. uMelt SM Dynamically Predicts Melt State Slider controls temperature and animates dissociation along amplicon

28. uMelt SM Prediction Matches Melt Curve for CFTR Exon 13 100bp 200bp

29. Troubleshooting SYBR ® Green qPCR Assays Observation/Problem Possible Cause Solution Extra peaks in melt curves Primer dimers Decrease primer concentration Increase annealing temperature Redesign primers Contamination Template contaminated with gDNA (bacterial target amplification) DNA polymerase in master mix contaminated with bacterial DNA a. Run “– RT” control b. Treat RNA template with DNase I or design primers to span exons Try new master mix AT-rich subdomains causing uneven melting Assess amplicon using uMelt SM tool Run a gel to verify single product

30. Troubleshooting SYBR ® Green qPCR Assays Observation/Problem Possible Cause Solution Poor amplification Reagent missing from assay Repeat experiment Annealing temperature too low Increase annealing temperature Detection temperature needs adjustment Set temperature of detection to be below amplicon T m , but above T m of primer dimers Set detection reading at the annealing step Amplicon is too long Amplicons longer than 500 bp are not recommended. Adjust extension time, if necessary Enzyme is not activated Follow enzyme activation time based on master mix Template concentration too low Use template concentration up to 500 ng

31. Steps for Designing a Reliable Assay Know your gene. Determine how many transcripts are associated with that gene. Identify exons that are common or specific between the transcripts. Obtain a RefSeq accession number Use NCBI databases to identify exon junctions, splice variants, SNP locations Align related sequences. For splice-specific designs: Identify unique regions within which to design primers and probe Avoid sequence repeats Perform BLAST searches of primer and probe sequences. Ensure no cross reactivity with other genes within the species Ensure that primers are not designed over SNPs. Run the amplicon through the uMelt SM software to predict number of peaks.

32. Primer Design Criteria Melting temperature (T m ) Primer T m values should be similar ±2  C Normally ~60 – 62  C Length Aim for 18  30 bases GC content Do not include runs of 4 or more Gs GC content range of 35 – 65 % (ideal = 50 %) Sequence Avoid sequences that may create secondary structures, self dimers, and heterodimers (IDT OligoAnalyzer ® Tool ) Amplicon L ength Ideal amplicon size: 80–200 bp Design If measuring gene expression, design primers to span exon junctions Always perform a BLAST search of potential primer sequences and redesign if primer sequence is not target specific.

33. Primer Assays from IDT for Human, Mouse, and Rat

34. Conclusions Intercalating dye use in qPCR is inexpensive and flexible. Observing the DNA melt dynamics of the amplicon via dye binding can be a useful tool for distinguishing good data from bad. Take care when interpreting melt data due to the potentially complicated nature of melting. Before doing qPCR , get to know your gene and optimize assay and primer design. uMelt SM software is a useful online tool that can help you predict unexpected melt dynamics.

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