Mission trc hs 1

Manufactured by Merck Group

The MISSION® TRC-Hs 1.0 is a lab equipment product offered by Merck Group. It is a thermal cycling instrument designed for nucleic acid amplification techniques such as PCR (Polymerase Chain Reaction). The core function of this product is to precisely control the temperature cycling required for DNA/RNA amplification.

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MISSION TRC-Hs 1.0 and 1.5 libraries (2 citations)

3 protocols using mission trc hs 1

Pancreatic Cancer Cell Line Characterization and Manipulation

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Pancreatic cancer cell lines used in this study (BxPC-3 [#CRL-1687), SW1990 [#CRL-2172], SU.86.86 [#CRL-1837], PANC-1 [#CRL-1469], Hs 766T [#HTB-134], CFPAC-1 [#CRL-1918], MIA PaCa-2 [#CRM-CRL-1420], AsPC-1 [#CRL-1682], Panc 03.27 [#CRL-2549], HPAF-II [#CRL-1997], and Capan-2 [#HTB-80]) were purchased from ATCC and maintained in RPMI-1640 with 10% fetal bovine serum (FBS). The identity of each cell line was confirmed by DNA fingerprinting via short tandem repeats at the time of mRNA and total protein lysate preparation using the PowerPlex 1.2 kit (Promega). Fingerprinting results were compared with reference fingerprints maintained by the primary source of the cell line.
Transient knockdown of CES2 was performed by transfecting the cells using the following siRNAs: siControl (Silencer Select Negative Control No. 1, Thermo Fisher Scientific) and siCES2 (s225041, s528; Thermo Fisher Scientific). Short-hairpin RNAs targeting human CES2 mRNA and cloned into the pLKO.1-puro vector were obtained from the human library MISSION® TRC-Hs 1.0 (Sigma–Aldrich, TRCN0000046965). For overexpression, CES2 was cloned into the pLenti-C-Myc-DDK-IRESPuro (OriGene) vector, and an empty vector was used as a control. Lentiviral infections were conducted using 293LTV cells (Cell Biolabs, Inc.).

Chen Y., Capello M., Rios Perez M.V., Vykoukal J.V., Roife D., Kang Y., Prakash L.R., Katayama H., Irajizad E., Fleury A., Ferri-Borgogno S., Baluya D.L., Dennison J.B., Do K.A., Fiehn O., Maitra A., Wang H., Chiao P.J., Katz M.H., Fleming J.B., Hanash S.M, & Fahrmann J.F. (2021). CES2 sustains HNF4α expression to promote pancreatic adenocarcinoma progression through an epoxide hydrolase-dependent regulatory loop. Molecular Metabolism, 56, 101426.

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Knockdown of HOXC6, MARK4, and PRNP in HCT116 and SW480 Cells

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HOXC6, MARK4, and PRNP-specific small interfering RNAs (siRNAs) and a negative control siRNA (si-NC) were synthesized by Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). The siRNA sequences were the following:

si-HOXC6 #1, 5′-UCCUACUUCACUAACCCUU[dT][dT]-3′;

si-HOXC6 #2, 5′-CCUCAAUUCCACCGCCUAU[dT][dT]-3′;

si-MARK4 #1, 5′-GCAUCAUGAAGGGCCUAAA[dT][dT]-3′

si-MARK4 #2, 5′-CCAUCUACCUUGGGAUCAA[dT][dT]-3′;

si-PRNP #1, 5′-GCGUCAAUAUCACAAUCAA[dT][dT]-3′;

and si-PRNP #2, 5′-GCCUAUUACCAGAGAGGAU[dT][dT]-3′.

The siRNAs were transfected into HCT116 cells using Lipofectamine RNAiMax (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA) at a final concentration of 50 nM. RNA was extracted 48 h after transfection. We also obtained three lentiviral vectors containing short hairpin RNAs (shRNAs) directed to ARC and an empty vector (pLKO.1 puro) from the MISSION TRC-Hs1.0 library (Sigma-Aldrich; Merck KGaA). The four lentiviral vectors were co-transfected into 293FT cells with VSVG and PAX2 plasmids using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific). The supernatant containing the lentivirus was collected 48 h after transfection and added to SW480 cells with 5 μM polybrene. Stable colonies were selected clonally with 5 μg/ml puromycin for 14 days, and sh-negative control (sh-NC), sh-ARC #1, #2, and #3 were established.

Ishikawa S., Nishida N., Fujino S., Ogino T., Takahashi H., Miyoshi N., Uemura M., Satoh T., Yamamoto H., Mizushima T., Doki Y, & Eguchi H. (2021). Comprehensive profiling of novel epithelial–mesenchymal transition mediators and their clinical significance in colorectal cancer. Scientific Reports, 11, 11759.

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Lentiviral Knockdown of HSulf-1 in OV202 Cells

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HSulf-1-targeting ShRNA1 (Sh1), which targets the 3′-untranslated region (UTR), was cloned into lentiviral vector pLKO.1-puro as described previously.^{1 (link)} HSulf-1-targeting ShRNA2 (Sh2) and nontargeting control ShRNA (NTC shRNA) cloned into the lentivirus vector pLKO.1-puro were chosen from the human library (MISSION TRC-Hs 1.0) and purchased as glycerol stock from Sigma-Aldrich (St. Louis, MO). Transfection with Fugene (Roche, South San Francisco, CA) was performed according to the manufacturer's instructions. Transfected cells were selected with 1 lg/ml puromycin. Two populations of transduced cells (OV202 Sh1 and Sh2) were generated instead of clonal lines generated from individual colonies.

He X., Khurana A., Roy D., Kaufmann S, & Shridhar V. (2014). Loss of HSulf-1 expression enhances tumorigenicity by inhibiting Bim expression in ovarian cancer. International journal of cancer. Journal international du cancer, 135(8), 1783-1789.

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