Digital PCR was performed using the QuantStudio 3D PCR system (Applied Biosystem) according to the manufacturer's instructions. Reactions were prepared using specific primers and probes for the target detection as reported in Supplementary Table S1. The equivalent amount of 100 ng of retrotranscribed RNA was used for the absolute quantification of SNORA67-EIF4A1 species and H/ACA EIF4A1 mRNA; results were normalized against the GUS housekeeping gene.
Quantstudio 3d pcr system
Manufactured by Thermo Fisher Scientific
The QuantStudio 3D PCR system is a digital PCR instrument designed for high-precision, sensitive, and quantitative nucleic acid analysis. The system utilizes a microfluidic chip-based platform to perform digital PCR reactions, enabling accurate and reliable quantification of target sequences.
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4 protocols using quantstudio 3d pcr system
1
Absolute quantification of SNORA67-EIF4A1
2
Absolute Quantification of snoRTs
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Digital PCR was performed using the QuantStudio 3D PCR system (Applied Biosystem) according to the manufacturer’s instructions. Reactions were prepared using specific primers and probes for the target detection as reported in Additional file 3 : Table S2. The equivalent amount of 100 ng of retrotranscribed RNA was used for the absolute quantification of 3′ EIF4A1 snoRTs fragment and full-length EIF4A1 snoRT; results were normalized against the GUS housekeeping gene.
3
Digital PCR Quantification of MYD88 L265P
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The digital PCR primer/probe mix of MYD88 L265P was obtained from Bio‐Rad (Refseq NC_000003.11, NG_023225.1, NT_022517.18, and NG_016954.1). For the detection and quantification of nucleic acids, the QuantStudio 3D PCR system (Thermo Fisher Scientific) was used. We mixed 7.5 μL of 2× Quant Studio 3D digital PCR Master Mix (Thermo Fisher Scientific), 0.75 μL of 20× target and wild probe (MYD88) (Bio‐Rad), and 10 ng of DNA to prepare a total volume of 15 μL. A 14.5 μL aliquot of the mixture was transferred to the Sample Loading Blade, and applied to the QuantStudio 3D Digital PCR 20K Chip using the Chip Loader. Amplification was performed using the Dual Flat Block Gene Amp PCR System 9700 (Thermo Fisher Scientific). In the analysis, the specific signal of the MYD88 L265P mutation was detected as a FAM signal, and the wild‐type signal was detected as a VIC signal. In this system, the fraction of the MYD88 L265P mutation dye signal over the total dye signal from the target gene (termed “Target/Total” within the QuantStudio™ 3D AnalysisSuite™ Software, Thermo Fisher Scientific) in the sample was calculated using the following formula:
The thresholds used were those automatically determined by the system and were readjusted manually only when the thresholds clearly crossed the cluster.
The thresholds used were those automatically determined by the system and were readjusted manually only when the thresholds clearly crossed the cluster.
4
Quantitative RT-PCR Analysis of Cardiac Biomarkers
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Reverse transcription-quantitative (RT-q)PCR was performed to detect mRNA levels of miR-155, atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and B-myosin heavy chain (B-MHC). Primer3 Plus (http://www.primer3plus.com/cgi-bin/dev/primer3plus.cgi ) was used to design the primers. According to the manufacturer's protocol, mouse myocardial tissues or isolated rat ventricular myocytes (NRVMs, 1×106) were treated with TRIzol® (cat. no. 15596026; Thermo Fisher Scientific, Inc.) to extract total RNA. According to the manufacturer's protocol, reverse transcription of RNA was then performed using a miRNA reverse transcription kit (cat. no. 4366597; Thermo Fisher Scientific, Inc.) and PrimeScript RT-PCR kit (cat. no. RR014A; Takara Bio, Inc.). The cDNA was used as a template and RT-qPCR was performed on QuantStudio 3D PCR system (Thermo Fisher Scientific, Inc.) with QuantStudio 3D AnalysisSuite (Thermo Fisher Scientific, Inc.). qPCR thermocycling conditions were as follows: Denaturation at 94°C for 5 min, 37 cycles of 95°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec. All reactions were repeated three times. U6 and GAPDH were the internal controls. The relative expressions of miR-155, ANP, BNP and B-MHC were expressed as a function of cycle quantification (Cq) and analyzed by the 2−ΔΔCq method (27 (link)). The primers are listed in Table I .
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