Northern Blot Analysis of piRNA Expression

Northern blot for piR-1 and piR-2 was performed as described previously^{36 (link)}. BmN4 total RNA was resolved by 15% PAGE containing 7 M urea, transferred to Hybond N + membranes (GE Healthcare), and hybridized to 5′-end labeled antisense probes detecting piR-1 (5′-GTTCGAAACCAATCCGTTAGTTTTTGA-3′), piR-2 (5′-GCCGCAGACAGCAAATTCTCATGCTTTT-3′), and Bombyx 5 S rRNA (5′-GCTTGACTTCGGTGATCGGACGAGAAC-3′). Typhoon 9400 and ImageQuant ver. 5.2 (GE Healthcare) were used for storage phosphor autoradiography visualization, and ImageJ was used for band quantifications.

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Honda S., Loher P., Morichika K., Shigematsu M., Kawamura T., Kirino Y., Rigoutsos I, & Kirino Y. (2017). Increasing cell density globally enhances the biogenesis of Piwi-interacting RNAs in Bombyx mori germ cells. Scientific Reports, 7, 4110.

Publication 2017

Hybond N + membranes Typhoon 9400 ImageQuant ver. 5.2

Autoradiography Bombyx Membranes Northern blot Phosphor Rrna Typhoon Urea

Corresponding Organization :

Other organizations : Thomas Jefferson University, Cedars-Sinai Medical Center

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Variable analysis

independent variables

Northern blot for piR-1 and piR-2

dependent variables

Expression levels of piR-1 and piR-2

control variables

BmN4 total RNA
15% PAGE containing 7 M urea
Hybond N + membranes (GE Healthcare)
5'-end labeled antisense probes detecting piR-1, piR-2, and Bombyx 5 S rRNA
Typhoon 9400 and ImageQuant ver. 5.2 (GE Healthcare) for storage phosphor autoradiography visualization
ImageJ for band quantifications

controls

Positive control: Bombyx 5 S rRNA probe
Negative control: Not explicitly mentioned

Annotations

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