Blood samples of selected couples were collected and DNA was extracted. Low-coverage WGS results were analyzed and a total of 170 probes were designed to validate the genetics of micro-deletions and micro-duplications. Genomic DNA of 100-200 ng was denatured at 98˚C for 2 min in a 20-µl reaction containing 1.25 µl of 4X DNA lysis buffer, 2 µl of 10X Taq ligase buffer, 0.2 µl of Taq ligase, 1 µl of 20X probe mix and 10.55 µl of Mili-Q water. Ligations were performed on an ABI 2720 thermal cycler (Thermo Fisher Scientific, Inc.) using the following parameters: A total of 5 cycles of 95˚C for 30 sec; 60˚C for 3 h; 94˚C for 2 min; hold at 72˚C for 2 min until the addition of 20 µl of 2X EDTA. PCRs were performed to amplify the ligation products with the 20-µl reaction mixture containing 10 µl of 2Z PCR Master Mix (Thermo Fisher Scientific, Inc.), 1 µl of Primer Mix (Thermo Fisher Scientific, Inc.), 1 µl of the ligation product and 8 µl Milli-Q water. The PCR parameters were as follows: 95˚C for 2 min; 5 cycles of 94˚C for 20 sec, 62-1˚C/cycle for 40 sec and 72˚C for 1 min; 27 cycles of 94˚C for 20 sec, 57˚C for 40 sec, 72˚C for 1 min and 68˚C for 20 min, and a final hold at 4˚C. PCR products were diluted 5-fold prior to loading on the ABI 3730XL sequencer (Thermo Fisher Scientific, Inc.). Raw data were analyzed using GENEMAPPER 4.0 (Thermo Fisher Scientific, Inc.).
Primer mix
Manufactured by Thermo Fisher Scientific
Sourced in China, United States, Germany
Primer Mix is a pre-formulated solution containing oligonucleotide primers and other necessary components for performing polymerase chain reaction (PCR) amplification. The core function of Primer Mix is to provide a standardized and optimized starting point for PCR experiments, simplifying assay setup and ensuring consistent results.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Minelute pcr purification kit, by Qiagen (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Abi 3730xl sequencer, by Thermo Fisher Scientific (1 mentions)
Genemapper 4, by Thermo Fisher Scientific (1 mentions)
Abi 2720 thermal cycler, by Thermo Fisher Scientific (1 mentions)
Nuclease free water, by Thermo Fisher Scientific (1 mentions)
Viia7 thermocycler, by Thermo Fisher Scientific (1 mentions)
Viia7 software v1, by Thermo Fisher Scientific (1 mentions)
Qpcr master mix plus low rox, by Eurogentec (1 mentions)
First strand cdna synthesis kit, by GE Healthcare (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Applied biosystems veriti 96 well thermal cycler, by Thermo Fisher Scientific (1 mentions)
Ez1 advanced xl robot, by Qiagen (1 mentions)
Qiaamp dna investigator kit, by Qiagen (1 mentions)
Ez1 dna investigator kit, by Qiagen (1 mentions)
Dreamtaq green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Pikoreal96, by Thermo Fisher Scientific (1 mentions)
Rest software, by Qiagen (1 mentions)
6 protocols using primer mix
1
Validation of Micro-Deletions and Micro-Duplications
2
Optimized PCR Amplification Protocol
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PCRs were performed with a total reaction volume of 20 μL, which included 10 μL of Master Mix (Institute of Forensic Science, Ministry of Public Security, Beijing, China), 6 μL of Primer Mix (concentrations indicated in Table S1 ), 3 μL of nuclease-free water (Thermo Fisher Scientific), and 1 ng of template DNA except for sensitivity studies. The reaction mixture was kept at 95 °C for 5 min, followed by 28 cycles of denaturing at 95 °C for 30 s, annealing at 59 °C for 2 min, and extension at 72 °C for 2 min, with a final elongation step at 72 °C for 2 min. The PCR products were purified with the MinElute® PCR Purification Kit (Qiagen, Hilden, Germany).
3
Quantitative Real-Time PCR of Pluripotency and Lineage Markers
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Total RNA was extracted using the RNeasy Micro Kit (Qiagen), and reverse transcription was performed using the First-Strand cDNA Synthesis Kit (GE Healthcare) following the manufacturer's instructions. Quantitative real-time PCR was performed using the ViiA 7 thermocycler (Thermo Fisher Scientific) and ViiA 7 software v1.2 (Thermo Fisher Scientific), using the standard settings. Quantitative real-time PCR was carried out on 40 ng of cDNA, using the qPCR MasterMix Plus Low ROX (Eurogentec), and TaqMan gene expression assays for SOX17, FOXA2 (endoderm), SOX1, PAX6 (ectoderm), and GUSB (Housekeeping) or 1.8 μM primer mix (IDT) and 250 nM probe (Thermo Fisher Scientific) for POU5F1 (pluripotency) and UBC (housekeeping). Fold changes were calculated with the ddCt method and UBC and GUSB were used as endogenous control.
4
DNA Extraction and PCR Amplification
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The DNA extraction was performed automatically using an EZ1® Advanced XL robot (Qiagen, Hilden, Germany) together with an EZ1® DNA investigator Kit (Qiagen) or manually, using a QIAamp DNA Investigator Kit (Qiagen). DNA concentration was measured using a Qubit® 3.0 Fluorometer and a Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).
The PCR amplification was performed using 10 µL of primer mix (Thermo Fisher Scientific), 4 µL of 5X Ion Ampliseq™ HiFi Mix (Thermo Fisher Scientific), and 0.2–1 ng of input DNA in 6 µL of molecular grade water. An Applied Biosystems® Veriti® 96-well thermal cycler (Thermo Fisher Scientific) was used with the following cycling program: 99 °C 2 min; (99 °C, 15 s; 60 °C, 4 min) × 24 times. The PCR primer mix was kindly provided by Thermo Fisher Scientific.
The PCR amplification was performed using 10 µL of primer mix (Thermo Fisher Scientific), 4 µL of 5X Ion Ampliseq™ HiFi Mix (Thermo Fisher Scientific), and 0.2–1 ng of input DNA in 6 µL of molecular grade water. An Applied Biosystems® Veriti® 96-well thermal cycler (Thermo Fisher Scientific) was used with the following cycling program: 99 °C 2 min; (99 °C, 15 s; 60 °C, 4 min) × 24 times. The PCR primer mix was kindly provided by Thermo Fisher Scientific.
5
Mutational Analysis of KRAS and TP53
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Mutational analysis was performed for the oncogene Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) (Exon 2) and the tumor suppressor p53 (TP53) (Exon 5 and 7). Therefore 18 N1 primary tumors and 18 matched lymph node metastases were analyzed. Polymerase chain reaction (PCR) was performed under the following conditions: 12.5 μl DreamTaq Green PCR Master Mix (Thermo Fisher Scientific, Schwerte, Germany), 0.5 μl Primer-Mix (10×, final concentration 0.2 μM), 11.5 μl H2O and 0.5 μl Template DNA were mixed and the following program was executed in a thermal cycler: 95 °C for 3 min, 95 °C for 30 sec, melting temperature (Tm) for 30 sec, 72 °C for 20 sec, repeat 30 times and finally 72 °C for 5 min. The primer sequences and Tm can be found in Table 14 . The PCR products were purified according to the manufacturers’ protocol (MinElute PCR Purification Kit, QIAGEN, Hilden, Germany). The analysis was validated through positive and negative controls. Sanger sequencing was performed at GATC Biotech (Konstanz, Germany).
6
RT-qPCR analysis of gene expression
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RT-qPCR analyses were carried out with SYBR Green I protocol on PikoReal 96 (Thermo Fisher) [42 (link)]. A plate layout with duplicates for each cDNA and primer was created in advance. Suitable sequences of nucleotides were searched in PubMed and primers were blasted with PubMed tool. On 96-well plates a final volume of 20 µL/well (5 µL cDNA mix and 15 µL Primermix, Thermo Fisher) was applied. Every analyzed gene was normalized to the housekeeping genes β-ACTIN (ACTB) and Histocompatibility 3 (H3; Table 2 ). Ct-values of each sample were analyzed with PikoReal 2.2 software (Thermo Fisher). By using REST software (Qiagen, Hilden, Germany), significances between stressed groups and the control group were evaluated.
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