The detection of dsRNAs in raw and cooked plant organs (seed and leaves) was only tested using northern blotting which is based on probe affinity hybridization. This technique

is not quantitative or as discriminating as, for example, quantitative real time PCR (q-RT-PCR) or high-throughput sequencing of the small RNA pool ( buy VX-770 Heinemann et al., 2011). Northern blotting should have been used in conjunction with q-RT-PCR which can detect targets at even lower concentrations with high stringency because it uses small primers (e.g., around 15bp). The techniques complement one another because rearrangements that might produce false negative PCR results may be detected by northern blotting, and PCR is more sensitive. Moreover, the CTNBio risk assessment did not report on the possibility of unintended RNAs being transcribed from additional inserts in the form of truncated copies, which were detected, nor did it require confirmation of the sequence and structure of the intended and anticipated dsRNA molecules. To address these concerns, the UFSC researchers suggested that the Ulixertinib manufacturer intended dsRNA molecules should be confirmed and quantified, and safety testing

for adverse effects should include feeding studies. The safety assessment did include a rat feeding study, but for various reasons this was not considered to be satisfactory for testing the specific hypothesis of an adverse effect arising

from the dsRNA. In that study, Wistar rats were fed for 35 days. One group of 10 rats was fed raw transgenic pinto beans; a second group of ten rats was fed on conventional raw pinto beans; a third (control) group of ten rats was fed a casein-rich diet; and a fourth (control) group of four rats was fed a non-protein diet. However, only 3 rats per group were killed for the morphological, histological and biochemical analyses. The proponent did not perform any immunological analyses of pregnant rats or any second-generation rats as requested by CTNBio Normative Resolution Farnesyltransferase no 5 Annex III. They argued that “there was no scientific basis” to do so “since no alteration in animal weight gain was observed” (p. 106 Aragão and Faria, 2010b). Moreover, raw beans caused the death of many rats starting 20 days after the start of the experiment but the exact number of dead animals was not disclosed (Aragão and Faria, 2010b). It is well known that anti-nutritional factors common in beans, as for soybeans, are removed by cooking (Gupta, 1987), and these effects could have obscured any more subtle toxic or other potential effects of the dsRNA. In another study, rats were fed orally with a solution of 6 mg of total RNA extracted from leaves.