One step sybr primescript rt pcr kit 2 perfect real time

Both sides of SGs were dissociated from anesthetized rats at ZT0, ZT6, ZT12, ZT18, ZT24, ZT30, ZT36, ZT42, and ZT48, and total RNA was immediately extracted by a modified acid guanidium phenol-chloroform method following SG dissociation. For cell isolation, both sides of SGs were dissociated at ZT0 and at 6-h intervals from ZT0 to ZT24. Isolated acinar and duct-like cells were then subjected to total RNA extraction within 2 h after whole SG dissection. For these samples, sqPCR analysis (Thermal Cycler Dice, TaKaRa Bio) was performed. For all series of sqPCR analyses, the same quantity of total RNA (50 ng) was used. Expression level of the internal reference gene, β-actin, was measured using One Step SYBR® PrimeScript® RT-PCR Kit II (Perfect Real Time, TaKaRa Bio), using probes labeled with 6-carboxyfluorescein (6-FAM). The primers used are described in Supplementary Tables 1, 2. sqPCR analysis was performed using the comparative Ct method (2^−ΔΔCt, where Ct implies cycle threshold). This method was used to assess relative expression level of mRNA normalized to β-actin, using the Thermal Cycler Dice real time system software version 5.11.

The isolation of total RNA from 30 mg of liver tissue was performed with an SV Total RNA Isolation System (Promega GMBH, Mannheim, Germany), according to the manufacturer’s instructions. Total RNA quantity and purity were measured by spectrophotometry using BioDrop (BioDrop μLITE, BioDrop, Cambridge, UK). Reverse transcription and quantification of isolated RNA were performed by One-Step SYBR PrimeScript RT-PCR Kit II (Perfect Real Time, TaKaRa Bio Inc. Shiga, Otsu, Japan) according to the manufacturer’s manual, using a Stratagene MxPro3005 (Agilent Technologies, US and Canada) thermocycler. The oligonucleotides used for quantitative PCR were: CD36-F, 5′-gcctcctttccaccttttgt-3′, CD36-R, 5′-gattcaaacacagcatagatggac-3′, TGF-β1-F, 5′-aatacgtcagacattcgggaagca-3′, TGF-β1-R, 5′- tcaatgtacagctgccgtac-3′, IL6-F, 5′-tgatggatgcttccaaactg-3′, IL6-R, 5′-gagcattggaagttggggta-3′, β-Actin-F, 5′-actattggcaacgagcggtt-3′, β-Actin-R, 5′-tgtcagcaatgcctgggtac-3′, Cyclophilin-F, 5′-cttcttgctggtcttgccattcct-3′, Cyclophilin-R, 5′-ggatggcaagcatgtggtctttg-3′. Gene expression was relatively quantified using the ΔΔCt method (2^ΔΔCt) after it was normalized to the expression level of the housekeeping genes. [15 (link)] Data are presented as the fold change in the gene expression relative to the corresponding control group.

Total RNA was extracted from SR segments including the earlier stage of LRP (Part 3 in Supplementary Figure 4A) in 5-day-old seedlings using the NucleoSpin^® RNA Plant Kit (Macherey-Nagel, Germany) following the manufacturer’s instructions. Quantitative reverse-transcription PCR (qRT-PCR) was performed using the One-Step SYBR PrimeScript RT-PCR Kit II (Perfect Real-Time) (TaKaRa Bio, Japan) and StepOnePlus Real-Time PCR (Life Technologies, United States). The expression was normalized to that of OsUBQ5 (Os03g0234200). All primers are listed in Supplementary Table 1. The expression of OsDRP family genes was compared using the RNA-seq data in S- and L-type LRP with four biological replications (Kawai et al., 2022a (link)). The read counts were normalized by coding sequence length (per 1 kb) for all genes in each replicate to calculate transcript per million (TPM). The normalized read counts were further normalized by summing the normalized counts of all genes (per million reads). Means of the calculated TPMs of four biological replications were used to compare the expression levels between S- and L-type LRP. The significance of the difference between S- and L-type LRP was determined by Padj values calculated by DESeq2 (Love et al., 2014 (link)).