Establishing a rapid method to obtain pure and intact RNA molecules has revolutionized the field of RNA biology, enabling laboratories to routinely perform RNA analysis such as Northern blot, reverse transcriptase quantitative PCR and RNA sequencing. Here, we describe an application of the effective single-step method of RNA extraction (or guanidinium thiocyanate-phenol-chloroform extraction) applied to Leptospira species. This method is based on the powerful ability of guanidinium thiocyanate to inactivate RNases and on the different solubility of RNA and DNA in acidic phenol. This method allows one to reproducibly obtain total RNAs with high yield and integrity, as determined by capillary electrophoresis, suitable for the RNA sequencing technology.