An improved method for subtractive cloning of differentially expressed genes in higher plants by protective exonuclease digestion and discriminating PCR amplification

1997 ◽  
Vol 16 (7) ◽  
pp. 509-512
Author(s):  
T.-B. Wang ◽  
A. D. M. Glass
Keyword(s):  
Differentially Expressed Genes ◽  
Higher Plants ◽  
Pcr Amplification ◽  
Differentially Expressed ◽  
Improved Method ◽  
Subtractive Cloning ◽  
Exonuclease Digestion

scholarly journals Effects of duplicated mapped read PCR artifacts on RNA-seq differential expression analysis based on qRNA-seq

2018 ◽  
Author(s):  
Anna C. Salzberg ◽  
Jiafen Hu ◽  
Elizabeth J. Conroy ◽  
Nancy M. Cladel ◽  
Robert M. Brucklacher ◽  
...  
Keyword(s):  
Differentially Expressed Genes ◽  
Differential Expression Analysis ◽  
Pcr Amplification ◽  
False Positives ◽  
Differentially Expressed ◽  
Rna Seq ◽  
Gold Standard Method ◽  
Pcr Duplicates ◽  
Cdna Molecule

AbstractBest practices to handling duplicated mapped reads in RNA-seq analyses has long been discussed but a gold standard method has yet to be established, as such duplicates could originate from valid biological transcripts or they could be PCR-related artifacts. Here we used the NEXTflex™qRNA-SeqTM(aka Molecular Indexing™) technology to identify PCR duplicates via the random attachment of unique molecular labels to each cDNA molecule prior to PCR amplification. We found that up to 64.3% of the single end and 19.3% of the mouse paired end duplicates originated from valid biological transcripts rather than PCR artifacts. For single end reads, either removing or retaining all duplicates resulted in a substantial number of false positives (up to 47.0%) and false negatives (up to 12.1%) in the sets of significantly differentially expressed genes. For paired end reads, only the alignment retaining all duplicates resulted in a substantial number of false positives. This is the first effort to evaluate the performance of qRNA-seq using ‘real-world’ biomedical samples, and we found that PCR duplicate identification provided minor benefits for paired end reads but greatly improved the sensitivity and specificity in the determination of the significantly differentially expressed genes for single end reads.

scholarly journals Improved Method for Detecting Differentially Expressed Genes Using cDNA Indexing

BioTechniques ◽  
2000 ◽  
Vol 28 (5) ◽  
pp. 958-964 ◽  
Author(s):  
C.J. Shaw-Smith ◽  
A.J. Coffey ◽  
E. Huckle ◽  
J. Durham ◽  
E.A. Campbell ◽  
...  
Keyword(s):  
Differentially Expressed Genes ◽  
Differentially Expressed ◽  
Improved Method

Solid phase subtractive cloning in differentially expressed genes identification

2009 ◽  
Vol 37 (3) ◽  
pp. 1435-1438 ◽  
Author(s):  
Oscar F. D’Urso ◽  
Pietro I. D’Urso ◽  
Carlo Storelli ◽  
Santo Marsigliante
Keyword(s):  
Differentially Expressed Genes ◽  
Solid Phase ◽  
Differentially Expressed ◽  
Subtractive Cloning
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