Total RNA was extracted from samples comprising two whole grains using the Sigma-Aldrich Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, St Louis, MO) with the addition of a 6-min incubation with thermostable α-amylase (Megazyme, Wicklow, Ireland) in lysis buffer at room temperature prior to addition of β-mercaptoethanol (Betts et al., 2017 (link)). Following treatment with TURBO DNase-free (Ambion, Life Technologies, Waltham MA), cDNA synthesis was performed using SuperScript®III Reverse Transcriptase according to manufacturer's instructions (Life Technologies, Waltham, MA). Details of gene names, MLOCs and primer details are presented in Table S1 . QPCR primers were designed using Primer 3 software (Koressaar and Remm, 2007 (link)) and selected based on specificity as determined by blastn software (Table S1 ; Acland et al., 2013 (link)). qPCR was performed as described by Burton et al. (2008 (link)) with data normalised using the reference genes HvCyclophilin, HvGAPdH2, HvHSP70, and HvTubulin (Vandesompele et al., 2002 (link)).
Thermostable α amylase
Manufactured by Megazyme
Sourced in Ireland
Thermostable α-amylase is an enzyme that catalyzes the hydrolysis of starch and related polysaccharides. It is resistant to high temperatures and can function effectively in various industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Amyloglucosidase, by Megazyme (3 mentions)
Alcalase 2.5 l, by Novozymes (2 mentions)
Bacillus licheniformis, by Megazyme (2 mentions)
Amg 300 l, by Novozymes (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Turbo dnase free, by Thermo Fisher Scientific (1 mentions)
Amyloglucosidase, by Merck Group (1 mentions)
Termamyl 120 l, by Merck Group (1 mentions)
Alcalase 2.4 l, by Merck Group (1 mentions)
4 protocols using thermostable α amylase
1
RNA Extraction and qPCR Analysis of Whole Grains
2
Dietary Fiber Isolation and Analysis
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thermostable α-amylase Termamyl 120 L (EC 3.2.1.1, from Bacillus licheniformis, ≥500 U•mg -1 protein (biuret)), amyloglucosidase (EC 3.2.1.3, from Aspergillus niger, 260 U•mL -1 ), and protease Alcalase 2.4 L (EC 3.4.21.62, from Bacillus licheniformis, 2.4 AU•g -1 ; all from Sigma Aldrich, St. Louis, Missouri) were used for preparative dietary fiber isolation. A thermostable α-amylase (EC 3.2.11, from Bacillus licheniformis, 3000 U•mL -1 ) and an amyloglucosidase (EC 3.2.1.3, from Aspergillus niger, 36000 U•g -1 ; both from Megazyme, Bray, Ireland) were used in the approaches to analyze dietary fiber analysis and starch. In addition, a protease (EC 3.4.21.14, from Bacillus licheniformis, 9 U•mg -1 ; Megazyme, Bray, Ireland) was used in the analytical fiber approach. Driselase (from Basidiomycetes) was from Sigma-Aldrich (St. Louis, Missouri). All chemicals and reagents were of analytical grade and purchased from Carl Roth (Karlsruhe, Germany), Merck (Darmstadt, Germany), Sigma-Aldrich (St. Louis, Missouri), or VWR (Radnor, Pennsylvania).
3
Dietary Fiber Isolation from Apple Pomace
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Apple pomace was kindly provided by Herbafood Ingredients GmbH (Company of H&F-Group, Werder (Havel), Germany). Detailed information about dietary fiber contents, chemical composition, and polysaccharide structures is given in Schmid et al. [3] . All chemicals and reagents were of analytical grade and purchased from Carl Roth (Karlsruhe, Germany), Merck (Darmstadt, Germany), Sigma Aldrich (St. Louis, MO, United States), or VWR (Radnor, PA, USA). thermostable α-amylase Termamyl 120 L (EC 3.2.1.1, from Bacillus licheniformis, 120 KNU. g -1 ), the amyloglucosidase AMG 300 L (EC 3.2.1.3, from Aspergillus niger, 300 AGU g -1 ), and the protease Alcalase 2.5 L (EC 3.4.21.62, from Bacillus licheniformis, 2.5 AU g -1 from Novozymes (Bagsvaerd, Denmark) were used for preparative dietary fiber isolation. A thermostable α-amylase (EC 3.2.11, from Bacillus licheniformis, 3000 U mL -1 ) and an amyloglucosidase (EC 3.2.1.3 from Aspergillus niger) (Megazyme, Bray, Ireland) were used to analyze starch contents. These enzymes in addition to a protease (EC 3.4.21.14 from Bacillus licheniformis) (Megazyme, Bray, Ireland) were used for the analytical dietary fiber analysis excluding resistant starch.
4
Dietary Fiber Isolation from Apple Pomace
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Apple pomace was kindly provided by Herbafood Ingredients GmbH (Company of H&F-Group, Werder (Havel), Germany). Detailed information about dietary fiber contents, chemical composition, and polysaccharide structures is given in Schmid et al. [3] . All chemicals and reagents were of analytical grade and purchased from Carl Roth (Karlsruhe, Germany), Merck (Darmstadt, Germany), Sigma Aldrich (St. Louis, MO, United States), or VWR (Radnor, PA, USA). thermostable α-amylase Termamyl 120 L (EC 3.2.1.1, from Bacillus licheniformis, 120 KNU. g -1 ), the amyloglucosidase AMG 300 L (EC 3.2.1.3, from Aspergillus niger, 300 AGU g -1 ), and the protease Alcalase 2.5 L (EC 3.4.21.62, from Bacillus licheniformis, 2.5 AU g -1 from Novozymes (Bagsvaerd, Denmark) were used for preparative dietary fiber isolation. A thermostable α-amylase (EC 3.2.11, from Bacillus licheniformis, 3000 U mL -1 ) and an amyloglucosidase (EC 3.2.1.3 from Aspergillus niger) (Megazyme, Bray, Ireland) were used to analyze starch contents. These enzymes in addition to a protease (EC 3.4.21.14 from Bacillus licheniformis) (Megazyme, Bray, Ireland) were used for the analytical dietary fiber analysis excluding resistant starch.
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