LAUSR

to different qPCR master mix solutions has a negative effect on Ct values (Table E1). Finally, we compared the original and improved protocols with regard to both RNA isolation and RT-qPCR. A 7.4-fold increase in RNA yield was achieved by using the optimized RNA isolation protocol (Figure 1E). Moreover, optimization of the qPCR protocol resulted in significantly lower Ct values of GAPDH amplicons of all sizes compared with the original protocol (Table E2). In conclusion, we have developed a combined sputum processing/RNA extraction/RT-qPCR protocol to maximize the quantity and quality of RNA obtained from sputum, and to enhance detection of transcripts by qPCR. Successful elimination of bacterial DNA was shown to be a critical step in the optimization process. Our results should facilitate future gene-expression studies utilizing sputum. n