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. 2022 Aug 9:13:954976.
doi: 10.3389/fpls.2022.954976. eCollection 2022.

Optimization of quantitative reverse transcription PCR method for analysis of weakly expressed genes in crops based on rapeseed

Michael Moebes  1 , Heike Kuhlmann  1 , Dmitri Demidov  1 , Inna Lermontova  1
Affiliations

Optimization of quantitative reverse transcription PCR method for analysis of weakly expressed genes in crops based on rapeseed

Michael Moebes et al. Front Plant Sci. .

Abstract

Rapeseed (Brassica napus) is an allopolyploid hybrid (AACC genome) of turnip rape (B. rapa, genome: AA) and vegetable cabbage (B. oleraceae, genome: CC). Rapeseed oil is one of the main vegetable oils used worldwide for food and other technical purposes. Therefore, breeding companies worldwide are interested in developing rapeseed varieties with high yields and increased adaptation to harsh climatic conditions such as heat and prolonged drought. One approach to studying the mechanism of the epigenetically regulated stress response is to analyze the transcriptional changes it causes. In addition, comparing the expression of certain genes between stress- and non-stress-tolerant varieties will help guide breeding in the desired direction. Quantitative reverse transcription PCR (RT-qPCR) has been intensively used for gene expression analysis for several decades. However, the transfer of this method from model plants to crop species has several limitations due to the high accumulation of secondary metabolites, the higher water content in some tissues and therefore problems with their grinding and other factors. For allopolyploid rapeseed, the presence of two genomes, often with different levels of expression of homeologous genes, must also be considered. In this study, we describe the optimization of transcriptional RT-qPCR analysis of low-expression epigenetic genes in rapeseed, using Kinetochore Null2 (KNL2), a regulator of kinetochore complex assembly, as an example. We demonstrated that a combination of various factors, such as tissue homogenization and RNA extraction with TRIzol, synthesis of cDNA with gene-specific primers, and RT-qPCR in white plates, significantly increased the sensitivity of RT-qPCR for the detection of BnKNL2A and BnKNL2C gene expression.

Keywords: RNA isolation; RNA precipitation; RT-qPCR; cDNA synthesis; crops; low expression; qPCR sensitivity; rapeseed.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Comparison of different homogenization strategies in young seeds and buds. (A) Young seeds without TRIzol addition, (B) buds without TRIzol addition, (C) young seeds with addition of 100 μl TRIzol before homogenization, (D) buds with addition of 100 μl TRIzol before homogenization, (E) Impact of homogenization strategy on RNA integrity. Marker—Invitrogen gene ruler 1 kb + (Cat. No. SM0311); left—undegraded RNA (low yield) isolated without TRIzol addition before, 1 min of homogenization of frozen plant tissue; middle—undegraded RNA (high yield) isolated with addition of 100 μl TRIzol before, 1 min of homogenization of thawed plant tissue; right—degraded RNA (high yield) isolated without TRIzol addition before, 3 × 1 min of homogenization in order to reach comparable tissue disruption efficiency in comparison with + TRIzol samples.
FIGURE 2
FIGURE 2
Transformed cycle-threshold (2– ct) values of ACT7 and UBC21 transcripts in flower buds of B. napus (v. Palma). (A) Influence of sample homogenization without and with TRIzol™ addition on qPCR performance, light gray—samples without addition of TRIzol™ before homogenization, dark gray—samples with TRIzol addition. (B) Influence of RNA-isolation method on qPCR performance, light gray—samples isolated with TRIzol™- Reagent, dark gray—samples isolated with RNeasy® plant mini kit. (C) Influence of RNA precipitation on qPCR performance, light gray—unprecipitated RNA, dark gray—precipitated RNA. (D) Influence of additional RNA purification on qPCR performance, light gray –TRIzol™ RNA without additional purification, dark gray—additionally purified RNA using NucleoSpin® RNA Set for NucleoZOL after TRIzol™ isolation. Error bars: standard deviation of sample (N-1) N = 4.
FIGURE 3
FIGURE 3
Transformed cycle-threshold (2– ct) values of BnKNL2A and BnKNL2C transcripts in a cDNA-mix of all analyzed tissues of B. napus (v. Palma). (A) Influence of cDNA synthesis strategy on qPCR sensitivity, light gray—cDNA, synthetized with unspecific oligo (dT)18, dark gray–specific cDNA, synthetized with antisense oligonucleotides of the corresponding gene. (B) Influence of different qPCR plates on signal intensity, light gray—transparent plate, dark gray—white plate of the same manufacturer. Error bars: standard deviation of sample (N-1) N = 3.
FIGURE 4
FIGURE 4
Comparison of transformed cycle-threshold (2– ct) values of BnKNL2A and BnKNL2C transcripts in all analyzed tissues of B. napus (v. Palma). (A) cDNA, synthetized with unspecific oligo (dT)18 and measured in a transparent qPCR plate. (B) Specific cDNA, synthetized with antisense oligonucleotides of the corresponding gene and measured in a white qPCR plate. Error bars: standard deviation of sample (N-1) N = 3.
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