Cell cultures were lysed in RL lysis buffer (Norgen Biotek Corp., Thorold, ON, Canada). RNA was isolated from cells by using a Total RNA Purification kit (Norgen Biotek Corp., Thorold, ON, Canada). The quantification of the isolated RNA was determined by NanoDrop spectrophotometer (ND-1000, EuroClone, Milan, Italy). Reverse transcription was conducted with SuperScript III (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s instructions. qRT-PCR was performed with the use of the iQTM SYBR Green Super Mix (Bio-Rad Laboratories, Hercules, CA, United States) and specific primers (reported in Table 1 ). All reactions were performed in a 96-well format with the 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific, MA, United States). The relative quantities of specific mRNA were obtained with the use of the comparative Ct method and were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Maione et al., 2021 (link); Zuccolini et al., 2022 (link)). The expression of each target gene was assessed in triplicate (Ferrera et al., 2021 (link); Maione et al., 2021 (link)).
Rl lysis buffer
Manufactured by Norgen Biotek
Sourced in Canada
The RL lysis buffer is a reagent used in the process of cell lysis, which is the disruption of cell membranes to release the intracellular contents. It is a key component in various nucleic acid extraction and purification protocols.
Automatically generated - may contain errors
Lab products found in correlation
7900ht fast real time pcr system, by Thermo Fisher Scientific (4 mentions)
Superscript 3, by Thermo Fisher Scientific (4 mentions)
Iqtm sybr green supermix, by Bio-Rad (4 mentions)
Total rna purification kit, by Norgen Biotek (4 mentions)
Nanodrop spectrophotometer, by Euroclone (3 mentions)
Nanodrop, by Euroclone (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
4 protocols using rl lysis buffer
1
Quantitative Real-Time PCR Analysis
2
Quantitative RT-PCR Analysis of Gene Expression
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Cell cultures were lysed in RL lysis buffer (Norgen Biotek corp., Thorold, Canada). RNA was isolated from cells by using a Total RNA Purification kit (Norgen Biotek corp., Thorold, Canada). The quantification of the isolated RNA was determined by NanoDrop spectrophotometer (ND-1000, EuroClone, Milan, Italy). Reverse transcription was conducted with SuperScript III (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. qRT-PCR was performed with the use of the iQTM SYBR Green Super Mix (Bio-Rad Laboratories, Hercules, CA, USA) and specific primers (reported in Additional file 1 : Table S3). All reactions were performed in a 96-well format with the 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific, Massachusetts, USA). The relative quantities of specific mRNA were obtained with the use of the comparative Ct method and were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
3
Quantitative RT-PCR Expression Analysis
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Cell cultures were lysed in RL lysis buffer (Norgen Biotek corp., Thorold, Canada). RNA was isolated from cells by using a Total RNA Purification kit (Norgen Biotek corp., Thorold, Canada). The quantification of the isolated RNA was determined by NanoDrop spectrophotometer (ND-1000, EuroClone, Milan, Italy). Reverse transcription was conducted with SuperScript III (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. qRT-PCR was performed with the use of the iQTM SYBR Green Super Mix (Bio-Rad Laboratories, Hercules, CA, USA) and specific primers (reported in Table S5 ). All reactions were performed in a 96-well format with the 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific, MA, USA). The relative quantities of specific mRNA were obtained with the use of the comparative Ct method and were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
4
Quantitative RT-PCR Analysis of Cardiac Samples
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After collection, RV endomyocardial bioptic samples from HC donors and ACM patients were crushed by mechanical disruption using metallic-beads by a TissueLyser (Qiagen, Milan, Italy) in an appropriate amount of RL lysis buffer (Norgen Biotek corp., Thorold, Canada). Cell cultures were lysed in RL buffer. RNA, both from cells and tissues, was isolated by using a Total RNA Purification kit (Norgen Biotek corp., Thorold, Canada). The quantification of the isolated RNA was determined by a NanoDrop spectrophotometer (ND-1000, EuroClone, Milan, Italy). Reverse transcription was conducted with SuperScript III (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. qRT-PCR was performed with the use of the iQTM SYBR Green Super Mix (Bio-Rad Laboratories, Hercules, CA, USA) and specific primers (reported in Table S5 ). All reactions were performed in a 96-well format with the 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific, Massachusetts, USA). The relative quantities of specific mRNA were obtained with the use of the comparative Ct method and were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
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