The radiolabelled RNA was analysed by denaturing gel electrophoresis (7.5 M urea and 8% polyacrylamide) with RNA markers (T3 and T7 RNA Pol body-labelled run-off transcripts).
The labelled heteroduplexes were run on a non-denaturing gel (LongRanger gel mix, Cambrex, Mt. Waverly, VIC, Australia) on an ABI PRISM 377 DNA Sequencer (Applied Biosystems, Foster City, CA, USA), where each heteroduplex displays a characteristic mobility.
Amplifying the CDR3 region of the Ig gene using labelled primers and running the subsequent products on a high-resolution sequencing gel produces a characteristic spectratype, representing the distribution of different CDR3 sizes in the sample population.
Labelled PCR products were run in a 3130 Genetic Analyzer (Applied Biosystems).
These labelled products were then run on an ABI 310 sequencer (Applied Biosystems).
Labelled fragments were then run on an Abi-prism 310 Automated DNA Sequencer (Perkin Elmer Applied-Biosystems) and analysElmer Applied-Biosystemsis Softwand 2.0.
Labelled PCR products were run together with the internal size standard GeneScan ROX400 on an ABI 3110 (Perkin Elmer, Biosystems), and individuals were genotyped using Genescan Analysis software and Genotyper software (Perkin Elmer, Biosystems).
The FAM-labelled PCR products were run on the ABI Prism 3100 Genetic Analyzer capillary system (Applied Biosystem, Foster City, CA) using POP6 as separation matrix, filter set D for the detection of fluorescent signals, and ROX500 as internal size standard.
At the start of the 7th week, patients underwent repeat measurement of TEE using the doubly labelled water protocol (which ran for 14 days to the end of week 8).
To assess differential sensitivity between isotopic and non-isotopic probes, the experiment was run with reversed labelled probe (i.e. Fto cRNA labelled with DIG and Pomc labelled with 35S).
Response units for each labelled protein concentration during the run are shown.
