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Troubleshooting Guide for LunaScript RT Master Mix (Primer-free) Kit (NEB #E3025)

Note: For additional assistance please refer to product FAQs at www.neb.com/E3025. 

PROBLEM

POSSIBLE CAUSE(S)

SOLUTION(S)

First Strand cDNA Synthesis Low cDNA yield Low quality of RNA

Insufficient quantity of starting material
  • Check the integrity of the RNA using denaturing agarose gel electrophoresis (1) or BioAnalyzer®
  • RNA should have a minimum A260/A280 ratio of 1.8 or higher. Ethanol precipitation followed by a 70% ethanol wash can remove most contaminants such as EDTA and guanidinium. Precipitation with lithium chloride can remove polysaccharides (1).
  • Phenol/chloroform extraction and ethanol extraction can remove contaminant proteins such as proteases (1)
  • Repurify RNA samples using column-based methods e.g., Monarch Total RNA Miniprep Kit
  • Use sufficient amount of input RNA

2 Step RT-qPCR

 Poor linearity

Poor RNA integrity

Inhibitors in the RNA templates

Too much cDNA added in the qPCR experiments
  • Ensure the purity and quality of the RNA templates
  • Repurify RNA samples
  • Add < 20% cDNA to the amplification reaction
Positive signals in the NRT reactions 
  DNA contamination in

the RNA templates
  • Perform DNase I treatment to remove DNA contaminants
  • Use qPCR primers spanning exons
Positive signals in the NTC reactions Contamination in the reagents used in the cDNA synthesis and/or qPCR steps
  • Follow good laboratory RT-qPCR practices
  • Replace the contaminated reagent(s)
2 Step RT-qPCR
 Low yield of RT-PCR product

Poor RNA integrity

Inhibitors in the RNA templates

Insufficient quantity of starting material

Suboptimal PCR reaction conditions
  • Ensure the purity and quality of the RNA templates
  • Repurify RNA samples
  • Use sufficient amount of input RNA
  • Optimize PCR reactions, e.g., primers, annealing temperature; use < 10% cDNA in the PCR reactions

 

References 

1 Sambrook, J. and Russel, D.W. (2001) Molecular Cloning: A Laboratory Manual (3rd Ed.) Cold Spring Harbor: Cold Spring Harbor Laboratory Press.