Characterization of amylolytic enzyme overproducing mutant of <i>Aspergillus luchuensis</i> obtained by ion beam mutagenesis

Current Practices for Reference Gene Selection in RT-qPCR of Aspergillus: Outlook and Recommendations for the Future

“…In total, this query returned 575 results which were manually curated to exclude those used to quantify fungal load, or those that did not specifically examine gene expression in Aspergillus , but that mentioned members of the genus in the abstract and with RT-qPCR studies in organisms other than Aspergillus . This allowed us to obtain information from 90 primary studies ( Table 1 , Table S1 ) [ 16 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , <...>…”

“…Overall, 15 species of Aspergillus were examined: A. aculeatus [ 34 , 35 ]; A. carbonarius [ 32 , 36 , 37 ]; A. cristatus [ 24 ]; A. fischeri [ 38 ]; A. flavus [ 28 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 ]; A. fumigatus [ 16 , 21 , 22 , 23 , 27 , 31 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 ]; A. luchuensis [ 26 , 75 ]; A. nidulans [ 30 , 76 , 77 , 78 ...…”

“…In our literature search, we found 17 reported usages of actin [ 16 , 21 , 23 , 25 , 26 , 34 , 42 , 43 , 60 , 75 , 77 , 78 , 79 , 89 , 91 , 96 , 108 ], 12 usages of beta-actin [ 22 , 24 , 44 , 45 , 61 , 76 , 90 , 97 , 103 , 104 , 105 , 107 ] and one reported usage of gamma actin [ 30 ] as reference genes. Unlike in the human genome, which contains about 20 copies of actin genes that make up six different isoforms of actin proteins [ 133 ], most fungi are known to contain only one actin gene [ 134 , 135 ].…”

“…Thus in total, actin was used in 30 studies examining gene expression in A. niger (3) [ 89 , 90 , 91 ], A. nidulans (4) [ 30 , 76 , 77 , 78 , 79 ], A. fumigatus (6) [ 16 , 21 , 22 , 23 , 60 , 61 ], A. flavus (4) [ 42 , 43 , 44 , 45 ], A. parasiticus (3) [ 103 , 104 , 105 ], A. cristatus (1) [ 24 ], A. oryzae (3) [ 25 , 96 , 97 ], A. luchuensis (2) [ 26 , 75 ], A. terreus (2) [ 107 , 108 ] and A. aculeatus (1) [ 34 ] ( Table 1 ). Of these 30 studies, seven studies provided an explanation for using actin under the conditions tested [ 16 , 45 , 75 , 78 , 89 , 103 , 105 ], although only six of these studies provided experimental validation of its expression stability under the specific conditions [ 16 , 30 , 77 , 103 , 105 , 107 ] ( Table 1 , Table S1 ).…”

“…Additionally, RT-qPCR is useful for detecting low-abundance transcripts, as the high annealing temperature used during RT-qPCR allows for highly specific primer binding to the target gene [ 20 ]. Indeed, RT-qPCR is often used to confirm the results obtained using other approaches [ 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ]. However, in order to accurately quantify gene expression using RT-qPCR, the normalisation of mRNA levels to validated reference genes is required [ 18 ].…”