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One μg of DNase-treated RNA was converted to cDNA using specific stem-loop RT primer (Additional file 5) and HiFi-MMLV cDNA kit mix (CWBIO, Beijing, China).
One μg of DNase-treated RNA was converted to cDNA using oligo dT primer and MMLV cDNA kit mix (TaKaRa, Dalian, China).
DNA was amplified in a total volume of 10 μl, including 1 μl (5 30 ng) of genomic DNA, 0.2 µM of each primer, 4.2 mM MgCl 2, and 1× Roche High Resolution Melting Master kit mix.
Reaction components were: 2.5 μL of ABI Prism® SNaPshot® multiplex kit mix (Applied Biosystems), 0.5 μL of 10X AmpliTaqGold® PCR buffer, 3 μL of multiplex PCR products, 2.5 μL of deionized water, and 1.5 μL of a stock solution of extension primers (an unbalanced stock solution contained ∼5 μM of each extension primer, see Table 1 for the exact values).
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For RNA extraction, filters were immersed in RLT buffer (from a Quiagen RNeasy kit) mixed with an equal amount of 0.1 and 0.5 µm glass beads and subsequently vortexed.
DNA was extracted from the nasal and oral specimens using 200 μL of lysis buffer (provided with the kit) mixed with 100 μL of Copan swab transport media.
In brief, 2.5 μL of RNA were placed in 25 μL of reaction volume containing 12.5 μL of QuantiFast Probe RT-PCR Kit Master Mix, 0.25 μL QuantiFast RT Mix, 8.5 μL water and 1.25 μL of primers.
In brief, 2.5 μL of cDNA were placed in 25 μL of reaction volume containing 12.5 μL of QuantiFast Probe RT-PCR Kit Master Mix, 0.25 μL QuantiFast RT Mix, 8.5 μL water and 1.25 μL of primers.
PCR reactions were carried out using a commercial kit (HotStar Taq Plus Mastermix kit, Qiagen) according to the manufacturer's instructions.
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CEO of Professional Science Editing for Scientists @ prosciediting.com