The source of SYBR green master mix determines outcome of nucleic acid amplification reactions.
BACKGROUND
Quantitative (q) PCR by amplification of nucleic acid with a fluorescent dye is widely used. Selection of adequate PCR reagents and devices is relevant to achieve reliable and consistent data. Our main objective was to test the robustness of different commercial SYBR green PCR mixes with respect to specificity and sensitivity of the PCR assay, across various PCR machines (Light Cycler 96, ViiA7) and amplification protocols. Herein, we applied PCR protocols for determining mRNA transcript levels, DNA copy numbers, and DNA genotype.


RESULTS
First, we set up 70 primer-based assays that targeted immune-related mRNA transcripts. Of the 70 assays 66 (94.3 %) resulted in a single melting curve peak, indicating specificity of the amplification, with PCR mixes from large vendors (Roche, ABI, Bio-Rad). But this was only seen when the PCR protocol that was indicated in the vendor's guidelines for each particular mix was applied. When deviating from the prescribed protocol, suboptimal melting curves were most often seen when using Roche SYBR green. With respect to PCR yields, the use of ABI mix more often led to lower Cq values. Second, we set up 20 primer-selective PCR assays to target different insertion-deletion and single nucleotide polymorphism regions throughout the genome. The variation in delta Cq between positive and negative DNA samples among the PCR assays was the lowest when using ABI master mix. Finally, the quality of high resolution melting (HRM) assays for DNA genotyping was compared between four commercial HRM PCR mixes (Roche, Bioline, PCR Biosystems, ABI). Only Roche and ABI mixes produced optimal clusters of melting profiles that clearly distinguished genotype variants.


CONCLUSIONS
The current results show a preference for the use of ABI mix when it comes to obtaining higher sensitivity in cDNA analysis and a higher consistency among assays in distinguishing DNA genotypes among different individuals. For HRM assays, it is advisable to use master mix from a relatively large vendor.
Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Bldg 1, E3-Q, Albinusdreef 2, 2333, Leiden, The Netherlands.
Jianxin
J
Yang
Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Bldg 1, E3-Q, Albinusdreef 2, 2333, Leiden, The Netherlands.
Berit
B
Kemps-Mols
Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Bldg 1, E3-Q, Albinusdreef 2, 2333, Leiden, The Netherlands.
Marijke
M
Spruyt-Gerritse
Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Bldg 1, E3-Q, Albinusdreef 2, 2333, Leiden, The Netherlands.
Jacqueline
J
Anholts
Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Bldg 1, E3-Q, Albinusdreef 2, 2333, Leiden, The Netherlands.
Frans
F
Claas
Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Bldg 1, E3-Q, Albinusdreef 2, 2333, Leiden, The Netherlands. M.Eikmans@lumc.nl.
Michael
M
Eikmans
D052160:Benzothiazoles; D004247:DNA / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D003959:Diamines; D005456:Fluorescent Dyes / Q000737:chemistry*; D060005:Genotyping Techniques / Q000295:instrumentation / Q000379:methods; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation / Q000379:methods*; D009696:Nucleic Acids / Q000737:chemistry / Q000235:genetics*; D009930:Organic Chemicals / Q000737:chemistry*; D011804:Quinolines; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D011933:Reagent Kits, Diagnostic / Q000145:classification; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D015203:Reproducibility of Results
D052160:Benzothiazoles; D017931:DNA Primers; D018076:DNA, Complementary; D003959:Diamines; D005456:Fluorescent Dyes; D009696:Nucleic Acids; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger; D011933:Reagent Kits, Diagnostic; C098022:SYBR Green I; D004247:DNA
Genomic DNA; High resolution melting; Performance; Quantitative PCR; Specificity; mRNA