Cell And Gene Therapy: Research, Development & Regulatory Guidance

Step Loop RT-PCR for Detection of Small Interfering RNA (siRNA) The recent publications described a novel used the novel method for the detection of siRNAs using a TaqMan®-based approach. This approach utilizes similar strategy that has been used for microRNA detection. The approach is illustrated in below.  In brief, the RT step occurs in the presence of a stem-loop RT primer that is complementary to the last 6–10 bases of the 3′ end of the antisense strand of the target siRNA. The stem-loop primer contains an additional universal sequence at the 5′ end that facilitates a TaqMan-based detection strategy in the subsequent qPCR step. As in the case of microRNA, the forward primer for qPCR is sequence-specific for the target siRNA. For sequence compositions that yield a low predicted melting temperature (Tm), the forward primer is designed as a tailed primer to help increase Tm. Stem Loop PCR for SiRNA Detection Step 1: Preparation of liver and plasma samples for the quanti...

High-Resolution MS (HRMS) for Quantitation of Oligonucleotides HRMS in full-scan mode provides selectivity and can improve the detection limit considerably.  Therefore, HRMS is especially advantageous for the analysis of oligonucleotides. The method achieved a 5–10 ng/ml lower limit of quantitation (LLOQ) for double stranded oligonucleotides with an 18–22-mer oligonucleotide in each strand using TripleTOF mass spectrometer system.   Materials   Chemicals such as 1,1,1,3,3,3-hexafluoro-isopropanol (HFIP), diisopropylamine (DIPA), tetrahydrofuran, ammonium acetate and ammonium bicarbonate, were purchased from Sigma-Aldrich Inc. (MO, USA). HPLC grade methanol and acetonitrile were obtained from Fisher Scientific (PA, USA). Clarity OTX lysis buffer and SPE cartridges were obtained from Phenomenex Inc. (CA, USA).  Preparation of stock & working solutions  Oligonucleotide solution was prepared in deionized water to give a final concentration of 2 mg/ml. IS st...