- The amount of protein loaded per lane is low (we estimate
~1–2μg of total protein/100< 100 μm islets), and therefore
signals tend to be weak, especially for pACC. Therefore, expo-
sure times tend to be long (5–20 min). - Other researchers have described that long incubation times
(4–24 h) at 20 C increase the yield of precipitated RNA.
Nevertheless, in our hands, the differences are minimal, if any,
and we therefore adjust this step just for convenience. - For DNase treatment, we recommend the use of DNase I,
amplification grade from Invitrogen (18068-015), following
manufacturer’s instructions. This step is absolutely required if
the RNA is going to be used for RNA-seq but can be skipped if
the samples are used for other applications, such as qPCR, if
adequate primers (spanning exon junctions) are used. - The experience is run in triplicate. We recommend performing
a minimum of three independent experiments with mouse
islets. With human islets, a much higher number of experi-
ments might be required, due to the variability in the response
to glucose that is normally observed between donors/batches. - These experiments can be performed in combination with
pharmacological AMPK activators, such as 991 or C13. In
that case, the compounds are added to the KRBH buffer for
the duration of the experiment, including the preincubation. - More than ten islets per condition can be used. This is espe-
cially advisable if these are small (< 100 μm). - It is critical to make sure that all the islets used for each
condition have been collected, with the use of a microscope.
References
- Organization WH (2016) Global report on
diabetes. Geneva - Hawley SA, Ford RJ, Smith BK, Gowans GJ,
Mancini SJ, Pitt RD, Day EA, Salt IP, Steinberg
GR, Hardie DG (2016) The Naþ/GLUCOSE
cotransporter inhibitor canagliflozin activates
AMPK by inhibiting mitochondrial function
and increasing cellular AMP levels. Diabetes
65(9):2784–2794.https://doi.org/10.2337/
db16-0058 - Coughlan KA, Valentine RJ, Ruderman NB,
Saha AK (2014) AMPK activation: a therapeu-
tic target for type 2 diabetes? Diabetes Metab
Syndr Obes 7:241–253.https://doi.org/10.
2147/DMSO.S43731 - Salt IP, Johnson G, Ashcroft SJ, Hardie DG
(1998) AMP-activated protein kinase is acti-
vated by low glucose in cell lines derived from
pancreatic beta cells, and may regulate insulin
release. Biochem J 335(Pt 3):533–539- Sun G, Tarasov AI, McGinty J, McDonald A,
da Silva Xavier G, Gorman T, Marley A, French
PM, Parker H, Gribble F, Reimann F,
Prendiville O, Carzaniga R, Viollet B,
Leclerc I, Rutter GA (2010) Ablation of
AMP-activated protein kinase alpha1 and
alpha2 from mouse pancreatic beta cells and
RIP2.Cre neurons suppresses insulin release
in vivo. Diabetologia 53(5):924–936.https://
doi.org/10.1007/s00125-010-1692-1 - Fu A, Eberhard CE, Screaton RA (2013) Role
of AMPK in pancreatic beta cell function. Mol
Cell Endocrinol 366(2):127–134.https://doi.
org/10.1016/j.mce.2012.06.020 - Kone M, Pullen TJ, Sun G, Ibberson M,
Martinez-Sanchez A, Sayers S, Nguyen-Tu
MS, Kantor C, Swisa A, Dor Y, Gorman T,
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