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Open Access Highly Accessed Research article

Identification and validation of reference genes for quantitative RT-PCR normalization in wheat

Anna R Paolacci, Oronzo A Tanzarella, Enrico Porceddu and Mario Ciaffi*

Author Affiliations

Dipartimento di Agrobiologia ed Agrochimica, Università della Tuscia, Via S. Camillo de Lellis, 01100 Viterbo, Italy

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BMC Molecular Biology 2009, 10:11  doi:10.1186/1471-2199-10-11

Published: 20 February 2009

Additional files

Additional file 1:

List of articles reporting the application of reference genes for qRT-PCR normalization in wheat. The list was obtained by a PubMed search from January 1996 to March 2008 and includes 26 articles reporting 16 reference genes.

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Additional file 2:

RT-PCR primers used in this research. Table showing UniGene clusters and linked TC sequences of the reference genes used in qRT-PCR analysis, their primer sequences and characteristics of the corresponding amplicons.

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Additional file 3:

UniGene clusters and corresponding TC sequences of 177 selected candidate reference genes of wheat. The file reports the transcription profiles of the genes corresponding to the UniGene clusters in six wheat tissues (flower, inflorescence, leaf, root, seed and stem) calculated as a value representing the number of transcripts per million (TPM). Mean value (Mv), Variance (V), Standard deviation (SD) and Coefficient of Variation (CV) were determined on the expression values (TPM) of the 177 Unigene clusters in the six wheat tissues. The highest and lowest expression values, the relative expression (percentage) in the six wheat tissues and the number of ESTs included in each UniGene cluster are also reported. The frequency (number of hosting libraries/total number of cDNA libraries) was determined for each TC or group of TCs linked to the 177 selected UniGene clusters.

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Additional file 4:

Classification of the 177 selected candidate reference genes of wheat on the basis of their putative functions. File showing the UniGene clusters and corresponding TC sequences of the 177 selected candidate reference genes of wheat and their putative functional annotation determined on the basis of their orthologous genes of rice and relative GO annotations.

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Additional file 5:

Level of expression of the 177 selected candidate reference genes determined on the basis of the number of EST sequences included in each UniGene cluster. File showing UniGene clusters and corresponding TC sequences of the 177 selected candidate reference genes of wheat sorted in ascending order on the basis of the number of EST sequences included in each UniGene cluster.

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Additional file 6:

UniGene clusters and corresponding TC sequences of ten genes of the actin and α-tubulin families. Gene transcription profile of each UniGene cluster in six wheat tissues (flower, inflorescence, leaf, root, seed and stem) is reported as a value representing the number of transcripts per million (TPM). Mean value (Mv), Variance (V), Standard deviation (SD) and Coefficient of Variation (CV) were calculated on the expression values (TPM) of the ten Unigene clusters in the six wheat tissues. The highest and lowest expression values, the relative expression (percentage) in the six wheat tissues and the number of ESTs included in each UniGene cluster are also reported. The frequency (number of hosting libraries/total number of cDNA libraries) was determined for each TC or group of TCs linked to the 177 selected UniGene clusters.

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Additional file 7:

Expression analysis by RT-PCR of the genes wcor14 and TaHSP101B. Agarose gels of RT-PCR products of wcor14 and TaHSP101B genes after 35 PCR cycles in six samples consisting of two temperature treatments (4°C and 33°C) for 24 and 48 h and their controls (LTC = low temperature controls; HTC = high temperature controls). The transcripts of the constitutively expressed gene encoding actin (UniGene cluster Ta54825) were amplified as control. M = part of the DNA molecular weight marker XIV (Roche), the most intense band is 500 bp in length.

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Additional file 8:

Specificity of qRT-PCR amplification. Figures A1–A3 report the dissociation curves of 42 selected reference genes showing single peaks, they were obtained from three technical replicates of 18 cDNA pools representing different tissues and developmental stages of wheat. Figures B1–B3: Agarose gels (2%) showing amplification of a specific PCR product of the expected size for each candidate reference gene.

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Additional file 9:

PCR efficiency and Ct values for 46 primer pairs and 24 tissue samples used in this research. The file shows PCR efficiency and Ct values of two biological replicates performed on 24 tissue samples; three technical replicates were carried out for each of the 46 primer pairs. The 24 tissue sample comprised: 1) Roots from plants with single shoot and three leaves unfolded (Feekes scale 1.3); 2) Shoots1 = single shoots and leaves from plants at Feekes scale 1.3; 3) Shoots2 = shoots at the beginning of tillering (Feekes scale 2); 4) Shoots3 = shoots from plants with formed tillers (Feekes scale 3); 5) Shoots4 = shoots at the beginning of erect growth (Feekes scale 4); 6) flag leaves at booting stage (Feekes scale 10); 7) stems at booting stage (Feekes scale 10); 8–10) Spikes1–3 = spikes collected at intervals of 10–12 days (three developmental stages: 15–20 mm, flag leaf unfolding and heading stage); 11–16) single floral organs (glumes, lemma, palea, lodicules, stamens and pistil) from fully emerged spikes (Feekes scale 10.5); 17–18) Seeds 1–2 = seeds collected 15 (medium milk stage) and 30 (hard dough stage) days after anthesis; 18–24) six samples (seedlings at Feekes scale 1.3) consisting of two temperature treatments (4°C and 33°C) for 24 and 48 h and their controls (LT = low temperature; HT = high temperature).

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Additional file 10:

Expression stability of the genes belonging to four families (actin, α-tubulin, translation elongation and ADP-ribosylation factors) evaluated by statistical analysis of raw Ct values obtained in five different data-set. ANOVA analysis and statistics determined on raw Ct values obtained by 18 primer pairs which amplified either specific or multiple (m) gene transcripts of four gene families (Actin, α-tubulin, translation elongation and ADP-ribosylation factors) in five different data-sets: 1) all 24 samples including tissues, developmental stages and temperature treatments; 2) 18 samples relative to different tissues and developmental stages; 3) six samples consisting of two temperature treatments (4°C and 33°C), each for 24 and 48 h and their controls; 4) six samples relative to the single floral organs from fully emerged spikes; 5) six samples referring to vegetative tissues and developmental stages (shoots, stems and leaves).

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Additional file 11:

Ranking of the expression stability of the reference genes as calculated by geNorm in four data sets. geNorm output charts of M values for the 32 selected reference genes in four data sets: (A) = 18 tissues and developmental stages; (B) = six samples consisting of two temperature treatments (4°C and 33°C) for 24 and 48 h and their controls; (C) = six floral organs from fully emerged spikes; (D) = six vegetative tissues and developmental stages (shoots, stems and leaves).

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Additional file 12:

Determination of the optimal number of control genes for accurate normalization calculated on the basis of pair-wise variation (V) analysis in four data sets. geNorm output charts of V values for the 32 selected reference genes in four data sets: (A) = 18 tissues and developmental stages; (B) = six samples consisting of two temperature treatments (4°C and 33°C) for 24 and 48 h and their controls; (C) = six floral organs from fully emerged spikes; (D) = six vegetative tissues and developmental stages (shoots, stems and leaves).

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