Brightgreen 2 qpcr mastermix

Total RNA was isolated from cultured cells using miRNeasy Mini Kit (Qiagen, Germany) and converted into complementary DNA using 5× All-In-One RT MasterMix (abm, Canada). qRT-PCR was performed in triplicate for each sample using BrightGreen 2× qPCR MasterMix (abm, Canada) and a LightCycler 96 Real-Time PCR detection system (Roche, USA) according to the manufacturer’s instructions. For qRT-PCR, KCNQ1OT1 primers (Sangon Biotech, China) were: 5’-TGCAGAAGACAGGACACTGG-3’ (sense) and 5’-CTTTGGTGGGAAAGGACAGA-3’ (antisense); SNHG1 primers (Sangon Biotech, China) were: 5’-GCCAGCACCTTCTCTCTAAAGC-3’ (sense) and 5’- GTCCTCCAAGACAGATTCCATTTT-3’ (antisense); GAPDH primers (Sangon Biotech, China) were: 5’-GCATCCTGGGCTACACTG-3’ (sense) and 5’-TGGTCGTTGAGGGCAAT-3’ (antisense). The 2-^△△Ct method was used to calculate the relative gene expression levels of KCNQ1OT1 and SNHG1, which were normalized to the corresponding GAPDH mRNA levels.

To analyze Fshr expression in mouse tissues, total RNA was purified from adult female tissues using the RNeasy Plus Micro kit (Qiagen) according to the manufacturer’s instructions. For reverse transcription PCR, genomic DNA removal and cDNA synthesis were performed using a Maxima H Minus First Strand cDNA-Synthesis kit (Thermo Fisher). PCR was performed using a MyTaq Red Mix kit (Bioline). Real-time PCR was performed using a SensiFAST SYBR No-ROX one-step kit (Bioline) with a CFX-96 real-time PCR detection system (Bio-Rad Laboratories) as described previously^{45 (link)}.
To examine gene expression in cultured cells, total RNA from cells was extracted in TRIzol (Life Technologies, USA; Catalog No. 15596026) following the manufacturer’s instructions. 200 ng of RNA per sample was used for reverse transcription. Quantitative PCR was performed on a Corbett Rotorgene 600 instrument (Corbett Life Science) using BrightGreen 2× qPCR MasterMix (ABM, Mastermix-S). Primers used in reverse transcription and real-time PCR are described in Supplementary Table 1.

Genomic DNA for AFB screening was extracted by heating the honey bee homogenate [26 (link)]. Briefly, 800 µL of DEPC-treated water was added to a whole-bee homogenate aliquot and centrifuged at 800× g for 10 min. 200 µL of suspension from each aliquot were incubated at 95 °C for 15 min with the lids open, then centrifuged at 5000× g for 5 min. The screening was performed using a SYBR Green-based qPCR for the amplification and detection of a region in the 16S rRNA gene of P. larvae [27 (link)]. Reactions were carried out using BrightGreen 2× qPCR MasterMix (abm, Richmond, BC, Canada) in 20 µL reactions containing 5 µL of supernatant from heated honey bee homogenate and 250 nM of each primer. qPCR amplifications and detections were performed using a LightCycler^® 480 System (Roche Diagnostics, Roche, Basel, Switzerland) with a program consisting of initial denaturation at 94 °C for 4 min, followed by 45 cycles of denaturation at 95 °C for 15 s and annealing and signal acquisition at 56 °C for 10 s [27 (link)]. All screenings involved no template negative controls and positive controls.

Total RNA was extracted using the TriPure reagent protocol (Roche Life Sciences, Mississauga, ON, Canada). Following extraction, the RNA was resuspended in 20 µL of RNAse/DNAse-free H₂O (Wisent) and concentration equalized. RNA was then reversed transcribed using the QuantiNova™ kit (Qiagen, Toronto, ON, Canada) according to the kit’s instructions. Transcript expression was determined by using primers obtained from PrimerBank, designed through NCBI PrimerBlast, or from previous published studies [31 (link)] (Supplementary Table S1). These primers were used in conjunction with the BrightGreen 2× qPCR mastermix (ABM, Vancouver, BC, Canada). The relative transcript expression was determined using the delta-delta Ct method [32 (link)] and normalized to the averages of β ACTIN and HSP-90. The efficiencies of all primers were validated before use. For quantification of viral RNA, infected cells underwent total RNA extraction as described above. Reverse transcription was initiated using the OneScript cDNA Synthesis Kit (ABM, Vancouver, BC, Canada) as per the kit instructions, using random hexamer primers. Viral RNA was assessed using previously published primers (Supplementary Table S1) and shown relative to HSP-90 and/or β ACTIN. TCID50s were determined as described [33 (link)].

We performed RNA extraction with the FavorPrep™ Tissue Total RNA Mini Kit (FATRK 001, Favorgen) as per the manufacturer’s instructions. cDNA was acquired from 100 - 500 ng of RNA using 5× All-in-One RT Mastermix (G485, abm) and diluted 5 times. Quantitative polymerase chain reaction (qPCR) was performed with BrightGreen 2×qPCR MasterMix (MasterMix-LR-XL, abm) on a Roche Applied Science LightCycler 96 machine. We normalized the expression of genes was normalized to 18S or ZC2HC1C. All primers are listed in Table S4.

Total RNA (RNeasy mini kit Qiagen Cat. N° 74104) was prepared from ~1000 young adult synchronized nematodes and used for cDNA synthesis (Applied Biological Materials Inc. Cat. N° G490) followed by quantitative real-time PCR (qRT-PCR). qRT-PCR was performed with the BrightGreen 2 × q-PCR Mastermix (Applied Biological Materials Inc. Cat. N° MasterMix-LR) starting at 95 °C for 2 min, followed by 40 cycles at 95 °C for 5 sec, 60 °C for 30 sec and 72 °C for 30 sec. Transcript levels were normalized to the internal control act-1 encoding the actin protein. The forward and reverse primer sequences utilized in this study were: apn-1: 5′-GCACATCCAGAAGACGCTGC-3′ and 5′-TCTACGGTAGTTCCAGGGCT-3′; exo-3: 5′-AGGAGCCTGACCTCGTTTTT-3′ and 5′-GTAGCCACCGTTCTTCTCTG-3′; nth-1: 5′-TTTCCAGTCAAACCAGAGAT-3′ and 5′-AAATCCAACAGGACACAAAA-3′; ung-1: 5′-TTCCGGACATGTTCCTCAAA-3′ and 5′-TTCATTGCCCGCGGGAACTT-3; act-1: 5′-TGCTGATCGTATGCAGAAGG-3′ and 5′-TAGATCCTCCGATCCAGACG-3′.