Genescan 3

Microsatellite instability analysis was performed using the BAT26 primer set [5’-TGACTACTTTTGACTTCAGCC-3’ sense and 5’-AACCATTCAACATTTTTAACCC-3’ antisense]. The sense primer was labeled with 6-FAM. PCR was performed in a final volume of 25 μL containing 1 × PCR buffer, 3.0 mmol/L MgCl2, 0.2 μmol/L dNTPs, 0.4 μmol/L of each primer, 2 U of Platinum Taq DNA Polymerase (Invitrogen, Waltham, MA, USA) and 50 ng of DNA. The thermal conditions were 94 °C/5 min followed by 40 cycles [94 °C/1 min, 50 °C/1 min and 72 °C/1 min] and a final extension at 72 °C/7 min. The dye-labeled PCR products were analyzed with a ABI PRISM 3130 Genetic Analyzer using Genescan 3.7 software (Applied Biosystems, Waltham, MA, USA) according to Hoang JM et al. (1997) [36 (link),55 (link)].
After electrophoresis, gels were dried at 80 °C and exposed to radiograph film. The band pattern was compared between tumorous and non-tumorous tissues for each patient. To avoid PCR artifacts, all positive tests were duplicated. Only cases with microsatellite alterations at 3 or more loci [≥30% frequency], only in neoplastic tissue, were ascribed to microsatellite instability.

Terminal restriction fragment length polymorphism (T-RFLP) analysis was carried out to investigate the changes of methanogenic community in rice paddy soil [25] –[27] (link). In this study, T-RFLP patterns were compared in control and cattle and swine manure-applied soil at 40 Mg ha⁻¹ during rice cultivation. The primers used for the PCR amplification were described above, except that the forward primer was labeled with 6-carboxyfluorescein (6-FAM). The PCR product was purified by a PCR purification kit (Qiagen, Valencia, CA) and then digested by restriction enzyme Sau96I [G’GNCC] (New England Biolabs, NEB, Beverly, MA) at 37°C for 3 hr. The terminal restriction fragments (T-RF) were purified with SigmaSpin^TM Post-Reaction Clean-Up Columns (Sigma, St. Louis, MO, USA) and analyzed by using an ABI 3730 capillary sequencer (Applied Biosystems, Foster City, CA, USA). The T-RFLP pattern was analyzed with Genescan 3.7 software (Applied Biosystems) using peak height integration of the different T-RFs. The percent fluorescence intensity for single T-RF was calculated by using total fluorescence intensity of T-RFs. The diversity indices of methanogen community were assessed by Shannon diversity index (H’) and Shannon evenness (E), which were calculated based on T-RFLP data according to the method of Egert et al. [28] (link).

Microsatellite instability (MSI) analysis was performed using the BAT25 and the BAT26 primer set.¹⁸ The sense primers were labeled with FAM. Polymerase chain reaction (PCR) was performed, and dye‐labeled PCR products were analyzed with an ABI PRISM 3100 Genetic Analyzer using Genescan 3.7 software (Applied Biosystems, Darmstadt, Germany). A total of 0.5 µL PCR product was mixed with 9.25 µL highly deionized formamide and 0.25 µL DNA Size Standard LIZ 500 (–250; Applied Biosystems). This mixture was denatured for 3 min at 95°C, immediately placed on ice, and separated using an ABI 3130 Genetic Analyzer. Results were analyzed using GeneMapper software (Applied Biosystems). Both tumor and normal tissue samples were analyzed. Samples were divided into two groups: those with one or more of the two markers displaying MSI and those with no instability (microsatellite stable).

This was confirmed using 21 microsatellite markers including those recommended by the International Society of Animal Genetics (Supplementary Table 1). Sheep blood samples and cell lines were submitted for PCR and fragment analysis to an independent genetic Company (VHL Genetics, The Netherlands). PCR amplification and fragment analysis was conducted in an ABI PRISM 3130XL automatic sequencer (Applied Biosystems), and the amplified fragments were classified using the GENESCAN 3.7 software (Applied Biosystems). We undertook a blinded pairing of cloned sheep and parental cells, and all clones matched their cells of origin for all 21 markers (CERVUS v.3.0 software70 (link)).