Total RNA from wild-type and Alpl knockout clones were extracted with the PureLink RNA Mini Kit (Thermo Fisher Scientific) 1 day after cells were plated, and complementary DNA was synthesized with SuperScript III reverse transcriptase (Thermo Fisher Scientific) from 1 μg of total RNA according to the manufacturer’s instructions. Polymerase chain reaction (PCR) was performed with the StepOnePlus Real-Time PCR System (Applied Biosystems, MA, USA) and Fast SYBR Green Master Mix (Applied Biosystems). The expression level of each gene was normalized to both reference gene (Hprt1) and wild type. The primer sets for each target genes were as follows: Alpl, NM_007431 (forward) 5′-ctccaaaagctcaacaccaatg-3′ and (reverse) 5′-atttgtccatctccagccg-3′; Runx2, NM_009820 (forward) 5′-tggcttgggtttcaggttag-3′ and (reverse) 5′-ggtttcttagggtcttggagtg-3′; Sp7, NM_130458 (forward) 5′-ggagaccttgctcgtagatttc-3′ and (reverse) 5′-g cagagagacacccacagaaac-3′; Sparc, NW_009242 (forward) 5′-ggatgggttctgctctcatatt-3′ and (reverse) 5′-cctctgctctggccttaaatag-3′; Spp1, NM_009263 (forward) 5′-ctttcactccaatcgtccctac-3′ and (reverse) 5′-cagaaacctggaaactcctagac-3′; and Hprt1, NM_013556 (forward) 5′-ggccagactttgttggatttg-3′ and (reverse) 5′-cgctcatcttaggctttgtatttg-3′.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.