Rna lysis buffer

As described previously (Böttner et al., 2010 (link)), cryosections from isopentane frozen tissue blocks (14 μm) were placed on slides coated with polyethylene naphtalate (1 μm membrane, Carl Zeiss MicroImaging GmbH, Göttingen, Germany) and regions of interest were visualized by ultra-rapid (30 s) staining with cresyl violet according to P.A.L.M. RNA Handling Protocols (Zeiss MicroImaging, Göttingen, Germany). By inverse light microscopy (Axiovert, Zeiss, Jena, Germany), the myenteric ganglia were identified, laser-microdissected and collected by laser pressure catapulting (P.A.L.M. Microlaser Technologies, Bernried, Germany) in the lid of 0.5 ml reaction tubes. From each sample 2 mm² ganglionic tissue was collected, dissolved in 200 μl RNA lysis buffer (PEQLAB, Erlangen, Germany) and stored at −70°C. Longitudinal and circular muscle tissue was collected under stereomicroscopic control from cryosections (20 μm) with excluding the myenteric ganglia by carefully dissecting the tissue with a scalpel. For each sample, the tissue of six cryosections were collected, dissolved in 200 μl RNA lysis buffer (PEQLAB, Erlangen, Germany) and storage was carried out at −70°C.

HEK 293 cells were transiently transfected with the pAW48 expression vector harboring either wild‐type or mutated TBX5 sequences. Two days later, cells were lysed with RNA lysis buffer (Peqlab, Erlangen, Germany). Total RNA was purified, using the peqGOLD total RNA kit (Peqlab) and reverse‐transcribed into cDNA with M‐MLV reverse transcriptase (Invitrogen). Expression of NPPA (NM_006172.3), TBX5 (NM_000192.3), β‐ACTIN (NM_001101.3), CX40 (NM_005266.6), IRX4 (NM_016358.2), HEY2 (NM_012259.2), ID2 (NM_002166.4), and TNNI3 (NM_000363.4) was evaluated on a LightCycler 1.5 (Roche Diagnostics) using the following conditions: activation of Taq polymerase for 15 min at 95°C followed by 40 cycles with 15 sec at 94°C, 20 sec at 60°C, and 20 sec at 60°C.

As described previously [22 (link)] full-thickness biopsies of the colonic wall were immediately frozen in isopentane and stored at -70°C until use. Cryosections (14 μm) were placed on membrane-coated slides (polyethylene naphtalate, 1 μm, Carl Zeiss MicroImaging GmbH, Göttingen, Germany) and regions of interest were visualized by ultra-rapid (ca. 30 s) staining with cresyl violet according to manufacturer´s instructions (P.A.L.M. RNA Handling Protocols, Zeiss MicroImaging, Göttingen, Germany). Myenteric ganglia were identified by inverse light microscopy (Axiovert, Zeiss, Jena, Germany), excised by LCM and collected by laser pressure catapulting (P.A.L.M. Microlaser Technologies, Bernried, Germany) in the cap of 0.5 ml reaction tubes. From each sample 2 mm² ganglionic tissue was collected, immediately dissolved in 200 μl RNA lysis buffer (PEQLAB, Erlangen, Germany) and stored at -70°C.