Piko thermal cycler

Manufactured by Thermo Fisher Scientific

Sourced in Finland

The Piko Thermal Cycler is a compact and efficient instrument used for DNA amplification and thermal cycling applications in molecular biology and genetic research laboratories. The device is capable of precisely controlling temperature profiles to facilitate various polymerase chain reaction (PCR) protocols.

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Thermo Scientific PIKO 96-well Thermal Cycler Thermal cycler

7 protocols using piko thermal cycler

Investigating circVAPA and miR-615-3p Expression

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Total RNA was extracted by Trizol (Invitrogen, USA). In each sample, 2μg RNA was used to synthesize cDNA as the templates of RT-qPCR using MMLV (Promega, Beijing, China). RT-qPCR was carried out in triplicate using a PrimeScript RT reagent kit (Takara, Japan) on the piko^TM Thermal Cycler (ThermoFisher, USA). Relative expression levels of genes were calculated using the 2^−ΔΔCt method. U6 and GAPDH were used as internal controls for miRNA and mRNA, respectively. The sequences of primers are presented in Table 1.Table 1

Primers of qRT-PCR

Gene	Primers
circVAPA	Forward	5ʹ-GTGTCTGGCAAGGAACACTA-3ʹ
circVAPA	Reverse	5ʹ-GGTGGAGAAGAGGGACAATAAG-3ʹ
miR-615-3p	Stem-loop RT primer	5ʹ- GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGGCUC -3ʹ
	Forward	5ʹ UUCUCCCUCUGGGUCC-3ʹ
	Reverse	5ʹ-GTGCAGGGTCCGAGGT-3ʹ
SMARCE1	Forward	5ʹ-ATGGCCTTAGCTTAGGCT-3ʹ
SMARCE1	Reverse	5ʹ-TTGGCAATGCCGTATTAGC-3ʹ
GAPDH	Forward	5ʹ-AGCCACATCGCTCAGACAC-3ʹ
GAPDH	Reverse	5ʹ-GCCCAATACGACCAAATCC-3ʹ
U6	Forward	5ʹ-GCTTCGGCAGCACATATACTAAAAT-3ʹ
U6	Reverse	5ʹ-CGCTTCACGAATTTGCGTGTCAT-3ʹ

, & Xu Q. (2020). The Potential Tumor Promotional Role of circVAPA in Retinoblastoma via Regulating miR-615-3p and SMARCE1. OncoTargets and therapy, 13, 7839-7849.

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RAPD Amplification Protocol for Genomic DNA

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The RAPD reaction was performed in 50 ll reaction mixes consisting of 200 ng of genomic DNA, 4 mM MgCl 2 , 1 lM of primer (NZYTech), 0.5 mM dNTP mix, 5% (v/v) dimethyl sulfoxide (DMSO, Roche), NZYTaq buffer and 2.5 U of NZYTaq (NZYTech). The amplification was performed in a Piko TM Thermal Cycler (ThermoScientific) as follows: 95 °C for 2 min, 45 cycles of 95 °C for 1 min, annealing at 34 °C for 1 min and extension at 72 °C for 2 min, followed by a final extension step of 72 °C for 5 min. The amplification products were loaded on 1.5% agarose gel in Tris-borate-EDTA (TBE) and run in the same buffer for 2.5 h at 100 V.

Cunha J.T., Ribeiro T.I., Rocha J.B., Nunes J., Teixeira J.A, & Domingues L. (2016). RAPD and SCAR markers as potential tools for detection of milk origin in dairy products: Adulterant sheep breeds in Serra da Estrela cheese production. Food chemistry, 211.

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RT-PCR Analysis of TP53 Variants

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RT-PCR assay was performed with Piko™ Thermal Cycler (Thermo Fisher Scientific Inc.) using a OneStep RT–PCR Kit (Qiagen GmbH) as described in the manufacturer's instructions. Gene-specific PCR primers (IDT&Bio-Science Kft., Hungary) were designed by Primer3 software (http://frodo.wi.mit.edu/) and their binding site was verified by NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Optimal Tm of each primer was set at 59°C. Amplification cycles were optimized for each gene according to different gene expressions. Then, RT-PCR reaction for wild type and mutant TP 53 cell lines was carried out for each gene with an identical amplification cycle using 0.5 μg of total RNA. Amplification products were separated on a 2% agarose gel stained with GR Safe Nucleic Acid Gel Stain (Excellgen). Results were visualized and recorded by using the Glite900 BW Gel Scanner (Pacific Image Electronics). The analysis was repeated three times. PCR primers, product size and amplification cycles are shown in Supplementary Table 2.

Harami-Papp H., Pongor L.S., Munkácsy G., Horváth G., Nagy Á.M., Ambrus A., Hauser P., Szabó A., Tretter L, & Győrffy B. (2016). TP53 mutation hits energy metabolism and increases glycolysis in breast cancer. Oncotarget, 7(41), 67183-67195.

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Genotyping of Nhlrc2 Knockout Mice

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After trypsinization, the ES cell pellets were lysed with 0.1 mg/ml proteinase K in lysis buffer (0.1 M Tris pH 8.5, 5 mM ethylenediaminetetraacetic acid, 0.2% sodium dodecyl sulfate [SDS], 0.2 M NaCl), and the DNA was isolated through ethanol precipitation. E6.5–E8.5 DNA was isolated using 40 μl of QuickExtract (Lucigen, WI) solution according to the manufacturer's instructions, and 1–5 μL of the lysate was used for PCR.
PCR was used to amplify a region in Nhlrc2, where the insertion of the targeting construct resulted in a new SacI digestion site, with the primers shown in Table 2. Phire Hot Start II Polymerase (Thermo Fisher Scientific, Waltham, MA) and a Piko Thermal Cycler (Thermo Fisher Scientific, Vantaa, Finland) were used according to the manufacturer's instructions. Sanger sequencing was performed to validate the PCR product as described previously (Hiltunen et al., 2020 (link)). The PCR product from E6.5–8.5 embryos was precipitated with NaCl (4 M, 1:10) and ethanol before digestion overnight at −20°C or for 30 min at −70°C. The PCR product was digested using the SacI restriction enzyme (Thermo Scientific, Vilnius, Lithuania) according to the manufacturer's instructions and run on 1.5% agarose gel (BioNordika, Helsinki, Finland). SYBR Safe DNA Gel Stain (Invitrogen, Carlsbad, CA) was used for detection.

Hiltunen A.E., Vuolteenaho R., Ronkainen V., Miinalainen I., Uusimaa J., Lehtonen S, & Hinttala R. (2022). Nhlrc2 is crucial during mouse gastrulation. Genesis (New York, N.y. : 2000), 60(3), e23470.

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Quantitative RT-PCR Gene Expression Analysis

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To evaluate gene expression by quantitative reverse transcription polymerase chain reaction (PCR), the cells were seeded in 12-well plates and allowed to grow for 12 hours. Later, the cells were exposed to different formulations. The cells were washed in PBS and lysed by TRIzol reagent (Thermo Fisher, USA) at 6 hours after treatment. Then, 1 μg of total RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA) on a Piko Thermal Cycler (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Quantitative PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems, USA) on a 7500 Real-Time PCR System (Applied Biosystems, USA). The gene expression levels were evaluated using Ct in all tested samples and normalized to β-actin as a reference gene. The primer sequences are listed in table S1.

Huang K.W., Hsu F.F., Qiu J.T., Chern G.J., Lee Y.A., Chang C.C., Huang Y.T., Sung Y.C., Chiang C.C., Huang R.L., Lin C.C., Dinh T.K., Huang H.C., Shih Y.C., Alson D., Lin C.Y., Lin Y.C., Chang P.C., Lin S.Y, & Chen Y. (2020). Highly efficient and tumor-selective nanoparticles for dual-targeted immunogene therapy against cancer. Science Advances, 6(3), eaax5032.

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B Cell Total RNA Isolation and cDNA Synthesis

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Total RNA was extracted from B cells using the RNeasy Mini kit (Qiagen, Germany) according to the manufacturer’s instructions and treatment with RNase-free DNase Set (Qiagen, Germany) was performed to avoid contamination of genomic DNA. RNA concentration and purity were determined with NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, USA). Total RNA was reverse-transcribed into cDNA using DyNAmoTM cDNA Synthesis Kit for qRT-PCR (Finnzymes, Finland) with Moloney murine leukemia virus (M-MuLV) reverse transcriptase, random hexamers (300 ng/μl) and 2X RT Buffer, according to the manufacturer’s instructions, performed on Piko Thermal Cycler (Finnzymes, Finland). The cDNA samples were stored at -20°C.

Moura R.A., Quaresma C., Vieira A.R., Gonçalves M.J., Polido-Pereira J., Romão V.C., Martins N., Canhão H, & Fonseca J.E. (2017). B-cell phenotype and IgD-CD27- memory B cells are affected by TNF-inhibitors and tocilizumab treatment in rheumatoid arthritis. PLoS ONE, 12(9), e0182927.

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Mitochondrial Gene Amplification in Pituophis kaibarae

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We used PCR (polymerase chain reaction) to amplify a partial fragment of the mitochondrial control region (CR), Cyt b (cytochrome b), and COI (cytochrome oxidase subunit I) from a total of 128 individuals in eight P. kaibarae populations (Table S1) using previously published primers (see Table S2 for detailed information), CR (L-Thr and H-12S; Takahashi and Goto 2001 (link)), Cyt b (PunCytBF and PunCytBR; Miya et al. 2001 (link)), and COI (HCO and LCO; Folmer et al. 1994 (link)). PCR was performed using a Piko Thermal Cycler (Finnzymes, Espoo, Finland) with a 25-μL reaction mixture containing 1 μL genomic DNA, 1X Taq buffer, 0.2 mmol/L dNTP, 0.25 μmol/L of each primer, and 2.5 unit of Prime Taq DNA polymerase (GenetBio, Daejeon, South Korea). Thermal cycling consisted of a preliminary denaturation step at 94°C for 10 min followed by 35 cycles of 94°C for 30 sec, 54–58°C for 30 sec, and 72°C for 30 sec and final extension at 72°C for 10 min. PCR products were purified using a Primeprep PCR Purification Kit (GenetBio) for direct sequencing. The amplified products were sequenced by Genotech (Deajeon, South Korea) on an ABI 3730XL DNA Analyzer (Applied Biosystems, Foster City, CA).

Bae H.G, & Suk H.Y. (2015). Population genetic structure and colonization history of short ninespine sticklebacks (Pungitius kaibarae). Ecology and Evolution, 5(15), 3075-3089.

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