Fig 1.
Qualitative analysis of RNA by agarose gel electrophoresis.
Denaturing agarose gel (1%) electrophoresis of total extracted RNA (2μL) stained with GelRed (Biotium, Fremont-CA–USA). For the positive control (C+), total RNA was extracted from soybean (Glycine max) leaves, and extractions using P. guajava leaves were performed in duplicate (R1 and R2).
Table 1.
Analysis of RNA yield and purity for different methods.
Table 2.
Comparison of RNA extraction methods.
Fig 2.
Qualitative analysis of RNA from different tissues.
(A) Denaturing agarose gel (1%) electrophoresis of total extracted RNA (2μL) stained with GelRed (Biotium, Fremont-CA–USA). Extractions using the CTAB2 protocol were done in duplicate (R1 and R2) for different tissues from two distinct genotypes of P. guajava, Cortibel RM and Paluma. (B) Quality analysis of RNA samples from Cortibel RM cultivar by TapeStation (Agilent Technologies, Santa Clara-CA–USA) using RNA ScreenTape assay, after DNase I treatment, and RIN (RNA Integrity Number) were calculated. The analyses were performed with two samples of each tissue: immature leaf, young leaf, mature leaf, root, and flower bud, respectively. (C) Quality check of cDNA from CTAB2 samples (Cortibel RM tissues) by amplification of histone H2A gene measured using SYBR Green RT-qPCR, and melting curve showing specific amplification products.
Table 3.
Analysis of RNA yield and purity using CTAB2 method.
Fig 3.
Quantitative analysis by RT-qPCR to compare CTAB methods.
Linear regression curves of the real-time reverse transcription polymerase chain reaction assay based on serial dilutions of cDNA (1.0, 0.5, 0.25, and 0.125 μL) synthesized from the same volume of RNA (5 μL) were used for all samples. The log of cDNA volume (μL) is indicated on the x-axis, whereas the corresponding Ct values are shown on the y-axis. Each dot represents the mean result of three replicates. The correlation coefficient (R2) and linear equation of the regression analysis are shown. Values obtained in this assay for each sample are represented in solid lines and ideal values in dotted lines, estimated based on the Ct values of the highest dilution, considering an efficiency of one hundred percent in each cycle.