Skip to main content

Table 2 Candidate reference genes tested and primer sequences

From: Validation of a set of reference genes to study response to herbicide stress in grasses

Reference gene1 (accession number)

Primer sequences (5'-3')2

Amplicon lenght (bp)

Tm (°C)

Manual threshold3

PCR efficiency (%)

Regression coefficient (R2)

Average Cq value

CYC

F: AGCTTTGAAGTTGGCAGTAG

      
 

R: GATCGCGTATTCATGGACTTTAG

Discarded (aspecific amplification)

   

SPS

F: CATTGCAAGAACTATTTGTCACG

      
 

R: GCAGAGATCAAATGGTTCAAATC

      

ACT

F: TGTGCTTGACTCTGGTGATG

220

58

    
 

R: TTCATAATCAAGGGCAACGTAAGC

  

Discarded (aspecific amplification)

 

RUBISCO

F: CATTATCAAGAAGGGCAAGATGTG

169

60

    
 

R: TGTTGTACATCCCTGGAAGTTG

      

GAPDH

F: GTATTGTTGAGGGACTGATGACC

182

57

0.047

92

0.999

23.31

(JN599100)

R: AGTAAGCTTGCCATTGAACTCAG

      

TUB

F: TACTGTGGTTGAGCCATACAATG

162

60

0.069

98

0.993

23.87

(JN599101)

R: GCAGAGATCAAATGGTTCAAATC

      

EF1

F: CAAGTACTACTGCACCGTCATTG

199

57

0.025

89

0.984

25.10

(JN599095)

R: GATCATCTGCTTCACTCCAAGAG

      

UBQ

F: GCAAGAAGAAGACCTACACCAAG

225

60

0.054

100

0.991

19.00

(JN599096)

R: CCTTCTGGTTGTAGACGTAGGTG

      

18S

F: GTCCAGACATAGGAAGGATTGAC

245

63

0.052

106

0.995

15.08

(JN599097)

R: GAACATCTAAGGGCATCACAGAC

      

25S

F: GCATGAATGGATTAACGAGATTC

165

63

0.098

95

0.998

17.00

(JN599099)

R: GGCTCCCACTTATCCTACAC

      

26S

F: GATAGCGTACAAGTACCGTGAGG

238

63

0.102

94

0.993

20.45

(JN599098)

R: GTTTCGGGTCAAATAGGAAGAAC

      
  1. 1CYC cyclophilin, SPS sucrose phosphate synthase, ACT actin, RUBISCO ribulose biphosphate carboxylase, GAPDH glyceraldehyde-3-phosphate dehydrogenase, TUB beta-tubulin, EF1 elongation factor 1α, UBQ ubiquitin, 18S 18S ribosomal RNA (nuclear gene), 25S 25S ribosomal RNA (nuclear gene), 26S 26S ribosomal RNA (mitochondrial gene)
  2. 2F: forward primer and R: reverse primer
  3. 3The threshold for fluorescence detection was set using the logarithmic amplification plot so that it is above the background fluorescence, below the linear region and at the beginning of the region of exponential amplification (i.e. the linear portion of the plot)