Opticon monitor 3

Absolute quantitation of a specific mRNA in samples was determined by RT-qPCR. Ovarian total RNA was reverse transcribed using Omniscript cDNA sysnthesis kit (Qiagen, Valencia, CT) according to manufacturer’s protocol. In vitro transcription of the partial clone of the gene using AmpliScribe™ T7 High Yield Transcription Kit (Epicenter, Madison, WI) was performed to produce gene-specific mRNA containing the qPCR primer and probe sequences, which was reverse transcribed using Omniscript cDNA sysnthesis kit (Qiagen, Valencia, CT) according to manufacturer’s protocol to produce cDNA for standards. Primers for the qPCR were designed to be internal to the cloned region of the gene specific cDNA. Quantitative PCR was performed using BMP2 specific qPCR primers (F: 5′-GACACCAGGTTAGTGAATCAGAACA-3′, R: 5′-TCTTGGAGACACCTGGCTTCTC-3′), β-Actin-specific qPCR primers (F: 5′-TGACCGAGCGTGGCTACAG-3′, R: 5′-CTTCTCTTTGATGTCACGCACAAT-3′) and gene-specific FAM-labeled Taqman probes (BMP2: 6-FAM-5′-TGCTGTGATGCGGTGGACTGCA-3′-BLACKHOLE, β-Actin: 6-FAM-5′-TCACCACCACAGCCGAGAGGGA-3′-BLACKHOLE), cDNA and HotStar Taq DNA polymerase kit (Qiagen, Valencia, CT) following manufacturer’s protocol in a Chromo 4 thermocycler-detector (MJ research, Canada) and analyzed by Opticon monitor 3 (Biorad, Hercules, CA).

Mature flowers and buds were dissected on three independent plants using micro-dissecting forceps. Carpels, stamens, and tepals were removed separately and immediately frozen in liquid nitrogen. Forceps were cleaned with 100% ethanol between each tissue and flower. RNA for qPCR was extracted using Plant RNA Reagent (Invitrogen™), treated with Ambion TURBO DNA-free™, and converted to cDNA using Invitrogen Superscript III, primed using oligo(dT)₂₀ and a random hexamer. CcActin was selected as a reference gene based on successful preliminary trials demonstrating stable expression across tissues. Primer sequences are provided in Supplementary Table S1. Forty cycles of PCR were performed using either a BioRad DNA Engine Thermocycler or a CFX Connect Real-Time PCR Detections System (185-5200). A melting curve was performed from 60 °C to 95 °C with readings taken at 0.5 °C intervals. Relative gene expression was quantified using an Opticon Monitor 3 and CFX Manager software (both BioRad Laboratories, Inc.). Ct values were exported to Microsoft Excel, and ∆Ct values were calculated by subtracting the Ct of the reference gene, actin. Each dataset was statistically analysed in Excel using a t-test.

Total RNA was extracted using an RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. First-strand cDNA was synthesized by reverse transcribing 1 µg of total RNA using a Protoscript M-MuLV Taq reverse transcriptase-polymerase chain reaction (RT-PCR) kit (New England BioLabs) according to the manufacturer’s instructions. For real-time PCR analysis, TaqMan Gene Expression Assays (Applied Biosystems) including Hs00898637_m1 for CLSPN, Hs00967506_m1 for CHEK1 and Hs03003631_g1 for 18S rRNA were used to detect gene expression levels. PCR mixture contained 2 µl of diluted cDNA (corresponding to 4 ng of RNA), 5 µl of 2× TaqMan Master Mix (Applied Biosystems) and 0.5 µl of gene-specific 20× TaqMan Gene Expression Assay in a final volume of 15 µl. Real-time PCRs were performed using Chromo 4 Real-Time PCR Detector and Opticon Monitor 3 (Bio-Rad Laboratories). The thermal cycling conditions were a 10-min denaturation at 95°C and followed by 40 cycles of 15 s at 95°C and 1 min 11 s at 60°C. Three replicates for each sample were analyzed using the 2^−ΔΔCT method (27 (link)).

cDNAs synthesis for hsa-miR-324-3p, hsa-miR-331- 3p and
hsa-miR-451 (internal control) were performed by miRCURY LNA™ Universal
RT microRNA PCR (Exiqon, Denmark), as indicated by the manufacturer, and
UniSp6, RNA Spike-in template was used as a positive control. cDNA
products were incorporated into a master mix composed of 10 pmol/μl of
hsa-miR-324-3p, hsa-miR-331-3p and
hsa-miR-451 DNA primers (Exiqon, Denmark) and 2 U of ExiLEN SYBR® Green
master mix (Exiqon, Denmark). 20 µl of RT reaction was diluted 20× and 4 µl of the diluted
cDNA was used in 10 µl polymerase chain reaction (PCR) amplification reactions. A
non-template control (NTC) was added to verify the specificity of the quantitative reverse
transcription PCR (qRT-PCR). Reactions of qRT-PCR were carried out using Opticon Monitor 3
(Bio-Rad Laboratories Inc., USA). All reactions were carried out in triplicate. Data of
qRT-PCR were assessed according to the 2^-ΔΔCT method. All specific amplicons
resulted from qRT-PCR was loaded and electrophoresed on 12% non-denaturing polyacrylamide
gel electrophoresis (PAGE) in 1X Tris/ Borate/EDTA(TBE) buffer along with 50 bp DNA ladder
(Thermo Fisher Scientific, USA) and visualized by silver staining.

We expressed Tat fused with EGFP in mammalian cells. Tat-EGFP expression in cells was confirmed by laser-induced fluorescence using an inverted fluorescence microscope (Olympus IX71, Olympus, Tokyo, Japan). A 488 nm laser line was used for green fluorescence. The laser was focused into the channel using a 20× objective. The fluorescence signal was collected by the same lens and optically filtered. We detected the excitation/emission wavelengths of the green filter set U-MWIBA3 EGFP shifts (BP460-495; BA510-550). For the refolding EGFP reporter assay, the EGFP fluorescence emission spectra of the HIV Tat protein fused with EGFP in lysates from HeLa cells were measured in PBS buffer using the Chromo4™ System (Bio-Rad, Hercules, CA, USA). The EGFP reporter refolding assay measurements were performed at 30 °C. EGFP’s fluorescence kinetics were analyzed using the Opticon Monitor 3 (Bio-Rad). The multi-mode microplate reader FlexStation 3 system (Molecular Devices, LLC., San Jose, CA, USA) was also used for the detection of the EGFP fluorescence kinetics. The HIV Tat-EGFP fusion proteins harvested or isolated from HeLa cell lysates were denatured in 6 M guanidine-HCl and 1 mM DTT for 20 min at 40 °C. Next, the denatured proteins were diluted 25-fold in refolding buffer containing 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, and 5 mM MgCl₂.

After RNA isolation using TRIZOL^® Reagent (Invitrogen) and genomic DNA digestion with a DNA free kit (Ambion), cDNA was synthesized using ThermoScript kit (Invitrogen) and random hexamers following the manufacturer’s instructions. The quantitative PCR was performed using the SYBR^® Green Jumpstart^™ Taq ReadyMix^™ (Sigma-Aldrich^®) or SensiMix^™ (Bioline) in a Chromo4 DNA engine (MRJ) with Opticon Monitor 3 (BioRad) software. A list of primers used throughout this study is shown in S1 Table.