Skip to main content

Table 3 Classification of failed PrimerBank primer pairs.

From: A comprehensive collection of experimentally validated primers for Polymerase Chain Reaction quantitation of murine transcript abundance

Type of failure

Reasons for failure

Number of primer pairs

% from total analyzed

QPCR failures:

QT

No amplification detected

1745

6.5%

Agarose gel failures:

G1

No band observed on gel

1619

6.0%

G2

Multiple bands observed on gel

2177

8.1%

G3

Wrong size band observed on gel

645

2.4%

G4

Faint band observed on gel

224

0.8%

GT

Failed based on gel analysis criteria (G1–G4)

4665

17.4%

Sequencing failures:

ST

Low sequence quality

2378

8.9%

BLAST failures:

B1

Sequences obtained did not match to the expected sequences

1217

4.5%

B2

Low match length between sequences obtained and the expected sequences

2732

10.2%

B3

Low % identity between sequences, expected sequences were not 1st matches

1074

4.0%

BT

Failed based on BLAST analysis criteria (B1–B3)

5023

18.7%

  1. 26855 primer pairs, corresponding to 27684 transcripts were tested by QPCR, agarose gel electrophoresis, sequencing and BLAST. 26854 primer pairs, corresponding to 27683 transcripts, were tested by agarose gel electrophoresis, sequencing and BLAST. QPCR failures: QT. Total number of primer pairs for which no amplification was observed using SYBR Green I detection. Agarose gel failures: G1. Primer pairs for which no band could be seen on the agarose gel. G2. Primer pairs for which two or more bands could be seen on the agarose gel. G3. Primer pairs for which one band of the wrong (unexpected) size could be seen. G4. Primer pairs for which a faint band could be seen. GT. Total number of primer pairs which failed based on our gel analysis criteria. Sequencing failures: ST. Total number of primer pairs for which no PCR product sequencing information was obtained (low sequence quality, sequence reads less than 20–30 bases). BLAST failures: B1. Primer pairs whose PCR product sequences obtained did not match to the expected sequence by BLAST. B2. Primer pairs whose PCR product sequences obtained did not match to at least 50% of the length of the expected sequence by BLAST (nearly all for sequence quality reasons). B3. Primer pairs whose PCR product sequences obtained did not match with at least 92% identity to the expected sequence by BLAST, and/or for which BLAST did not return the expected sequence or any known isoforms as the first match. BT. Total number of primer pairs which were not successful based on our BLAST analysis criteria.
Back to article page

BMC Genomics

ISSN: 1471-2164

Contact us