Total RNA was isolated from fresh peripheral blood samples of the Patient-1 and his unaffected parents by using the RNAprep pure Blood Kit (DP433, TIANGEN) followed by first-strand cDNA synthesis (KR106, TIANGEN). cDNA was then amplified by polymerase chain reaction (PCR) with specific NEK3 primers followed by gel extraction (DP209, TIANGEN) and Sanger sequencing. The primers used in PCR reactions are listed in Supplementary Table S1 .
Dp209
Manufactured by Tiangen Biotech
Sourced in China
The DP209 is a compact and versatile lab equipment designed for various scientific applications. It functions as a temperature-controlled incubator, offering precise temperature regulation and monitoring capabilities. The device is suitable for a wide range of laboratory tasks that require a stable and controlled environment.
Automatically generated - may contain errors
Lab products found in correlation
Kr106, by Tiangen Biotech (2 mentions)
Rnaprep pure blood kit, by Tiangen Biotech (1 mentions)
Rnasimple total rna kit, by Tiangen Biotech (1 mentions)
Fastfire qpcr premix sybr green, by Tiangen Biotech (1 mentions)
Digoxygenin, by Roche (1 mentions)
Pgm t vector, by Tiangen Biotech (1 mentions)
Premix taq, by Takara Bio (1 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (1 mentions)
R6827, by Omega Bio-Tek (1 mentions)
4 protocols using dp209
1
NEK3 Gene Sequencing from Blood
2
Nasal RNA Sequencing Analysis
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Total RNA was isolated from fresh nasal tissue samples of the patients and healthy volunteers using the RNAsimple Total RNA kit (DP419; TIANGEN BIOTECH, Beijing, China), followed by first-strand cDNA synthesis (KR106; TIANGEN). cDNA was then amplified by polymerase chain reaction (PCR) with specific TTC12 primers, followed by gel extraction (DP209; TIANGEN) and Sanger sequencing. RT‒qPCR was performed using FastFire qPCR PreMix (SYBR Green) (FP207, TIANGEN) on an ABI StepOnePlus instrument. The delta-delta-Ct (ddCt) algorithm was utilized to analyze the relative changes in gene expression by using GAPDH as the internal reference (housekeeping gene). Primers were designed using the Primer3Plus software to span exon-exon junctions and provided in Additional file 8 : Table S4.
3
Cryosectioning and In Situ Hybridization of Fish Eyes
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Before cryosectioning, the eyes of cave and surface fish were stored in 30% sucrose at 4 °C overnight. The sections were stained with hematoxylin and eosin (H&E). For ISH, RNA probes were generated using Roche digoxygenin from Sinocyclocheilus cDNAs. The following primers were used to clone otx5 probes in PCR reactions: otx5-F: 5′-TGTGGTTTAAGAACCGTCGTG-3′ and otx5-R: 5′-GAACTTCCAGGAGTTCTGGTC-3′, which amplifies the exon3 of otx5. PCR amplification products were recovered from the gels with a DNA purification kit according to the manufacturer’s instructions (DP209; Tiangen Biotech) and then cloned into the pGM-T vector (VT202; Tiangen Biotech) in E. coli DH5a. Six clones were sequenced for every sample using T7 and Sp6 universal sequencing primers. Sequence-verified clones were used to generate antisense probes using SP6/T7 enzymes. ISH was performed with the color visualized using NBT/BCIP as described previously (Meng et al. 2013b (link)).
4
Isolation and Characterization of PcNRAMP1 cDNA
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Total RNA from the roots of P. × canescens was isolated and purified using a plant RNA kit (R6827, Omega Bio-Tek, Georgia, USA) as suggested [60 (link)]. Subsequently, cDNA was synthesized using Primescript™ 1st strand cDNA synthesis kit (6210A, Takara, Dalian, China) according to the manufacturer’s protocol. The full-length cDNA of PcNRAMP1 was amplified by specific primers (Table S1 ). The reaction system of PCR consisted of 50 µL, including 1 μL of cDNA, 0.5 μL of each of the primer (10 μM), 25 μL of Premix Taq™ (RR902A, Takara, Dalian, China) and 23 μL of H2O. The PCR was performed at 95 °C for 3 min, followed at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min 30 s with 31 cycles, and extended at 72 °C for 5 min. The PCR product was purified by gel extraction kit (DP209, Tiangen Biotech Co., Ltd. Beijing, China) and cloned into the pMD19-T vector for sequencing.
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