Xpress syber green er qrt pcr super mix

Total RNA was isolated from 10⁶ ATII cells 24 h after isolation using RNeasy MiniKit (Qiagen, Hilden, Germany). Reverse transcription was performed on 0.8 µg to 1.3 µg total RNA using the SuperScript VILO cDNA synthesis kit according to manufacturer's protocol. The following validated QuantiTect primer assays (Qiagen, Hilden Germany) were used: HMBS, Rn_Hmbs_1_SG; Syt1, Rn_Syt1_2_SG; Syt-2, Rn_Syt2_1_SG; Syt3, Rn_Syt3_1_SG; Syt5, Rn_Syt5_1_SG; Syt6, Rn_Syt6_1_SG; Syt7, Rn_Syt7_1_SG; Syt-9, Rn_RGD:621169_1_SG; Syt10, Rn_Syt10_2_SG; complexin-1, Rn_Cpl1_1_SG; complexin-2, Rn_Cpl2_1_SG; complexin-3, Rn_Cpl3_1_SG; complexin-4, Rn_Cpl4_1_SG. Amplification was performed on a realplex2 mastercycler (Eppendorf, Hamburg, Germany) using the XPress Syber Green ER qRT-PCR super mix. Each reaction was carried out on cDNA from three or more independent isolations (cDNAs were used at 1-, 10- and 100-fold dilutions). Specificity of PCR reactions was confirmed by melting points analysis of PCR products. Realplex software (Eppendorf, Hamburg, Germany) was used for data acquisition and analysis. Correction for PCR performance as well as quantification relative to housekeeping gene HMBS was carried out as described (Pfaffl, 2001 (link)).

Total RNA was isolated from 10⁶ ATII cells directly after isolation or following 48 h of culture in MucilAir medium with an RNeasy MiniKit (Qiagen, Hilden, Germany). Reverse transcription was performed on 0.8 µg to 1.3 µg total RNA using the SuperScript VILO cDNA synthesis kit according to manufacturer's protocol and validated QuantiTect primer assays (Qiagen, Hilden Germany) Amplification was performed on a realplex2 mastercycler (Eppendorf, Hamburg, Germany) using the XPress Syber Green ER qRT-PCR super mix. Each reaction was carried out on cDNA from ≥three independent isolations (cDNAs were used at 1-, 10- and 100-fold dilutions). Specificity of PCR reactions was confirmed by melting points analysis of PCR products. Realplex software (Eppendorf, Hamburg, Germany) was used for data acquisition and analysis. Correction for PCR performance as well as quantification relative to housekeeping gene Hmbs was carried out as described previously (Pfaffl, 2001 (link)).