scholarly journals Strand-specific detection of overlapping transcripts via purification involving denaturation of biotinylated cDNA

BioTechniques ◽  
2020 ◽  
Vol 69 (2) ◽  
pp. 141-147
Author(s):  
Faizan Uddin ◽  
Madhulika Srivastava
Keyword(s):  
Expression Analysis ◽  
Gene Expression Analysis ◽  
High Sensitivity ◽  
Rt Pcr ◽  
Specific Detection ◽  
Specific Primers ◽  
Reverse Transcription Pcr ◽  
Alkaline Denaturation ◽  
Priming Activity ◽  
Overlapping Transcripts

Reverse transcription-PCR (RT-PCR) is the most widely employed technique for gene expression analysis owing to its high sensitivity, easy reproducibility and fast output. It has been conceived that priming RT reactions with gene-specific primers generates cDNA only from the specific RNA. However, several reports have revealed that cDNA is synthesized even without addition of exogenous primers in RT reactions. Owing to such self-priming activity, the signals from specific strands cannot be accurately detected and can confound the expression analysis, especially in context of overlapping bidirectional transcripts. Here, we demonstrate that purification of biotin-tagged cDNA in conjunction with alkaline denaturation can obviate the problem of background priming and enable accurate strand-specific detection of overlapping transcripts.

scholarly journals Maximizing RNA Yield from Archival Renal Tumors and Optimizing Gene Expression Analysis

2009 ◽  
Vol 15 (1) ◽  
pp. 80-85 ◽  
Author(s):  
Sean T. Glenn ◽  
Karen L. Head ◽  
Bin T. Teh ◽  
Kenneth W. Gross ◽  
Hyung L. Kim
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Reverse Transcription ◽  
Gene Expression Analysis ◽  
Rna Isolation ◽  
Proteinase K ◽  
Specific Primers ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Formalin-fixed, paraffin-embedded tissues are widely available for gene expression analysis using TaqMan ® PCR. Five methods, including 4 commercial kits, for recovering RNA from paraffin-embedded renal tumor tissue were compared. The MasterPure™ kit from Epicentre produced the highest RNA yield. However, the difference in RNA yield between the kit from Epicenter and Invitrogen’s TRIzol method was not significant. Using the top 3 RNA isolation methods, the manufacturers’ protocols were modified to include an overnight Proteinase K digestion. Overnight protein digestion resulted in a significant increase in RNA yield. To optimize the reverse transcription reaction, conventional reverse transcription with random oligonucleotide primers was compared to reverse transcription using primers specific for genes of interest. Reverse transcription using gene-specific primers significantly increased the quantity of cDNA detectable by TaqMan ® PCR. Therefore, expression profiling of formalin-fixed, paraffin-embedded tissue using TaqMan® qPCR can be optimized by using the MasterPure™ RNA isolation kit modified to include an overnight Proteinase K digestion and gene-specific primers during the reverse transcription.

scholarly journals A bead-based microfluidic approach to integrated single-cell gene expression analysis by quantitative RT-PCR

RSC Advances ◽  
2015 ◽  
Vol 5 (7) ◽  
pp. 4886-4893 ◽  
Author(s):  
Hao Sun ◽  
Tim Olsen ◽  
Jing Zhu ◽  
Jianguo Tao ◽  
Brian Ponnaiya ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Single Cell Level ◽  
Rt Pcr ◽  
Cell Level ◽  
P Gene ◽  
Cell Gene Expression ◽  
Cell Gene

Gene expression analysis at the single-cell level is critical to understanding variations among cells in heterogeneous populations.

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